Biomaterials 35 (2014) 4465e4476

Contents lists available at ScienceDirect

Biomaterials
journal homepage: www.elsevier.com/locate/biomaterials

Review

Biomaterials in co-culture systems: Towards optimizing tissue

integration and cell signaling within scaffolds
Kyle G. Battiston a,1, Jane W.C. Cheung a,1, Devika Jain a, J. Paul Santerre a, b, *
a
Institute of Biomaterials and Biomedical Engineering, University of Toronto, 124 Edward Street, Room 461, Toronto, Ontario, Canada M5G 1G6
b
Department of Biomaterials, Faculty of Dentistry, University of Toronto, 124 Edward Street, Room 464D, Toronto, Ontario, Canada M5G 1G6

a r t i c l e i n f o a b s t r a c t

Article history: Most natural tissues consist of multi-cellular systems made up of two or more cell types. However, some
Received 4 January 2014 of these tissues may not regenerate themselves following tissue injury or disease without some form of
Accepted 12 February 2014 intervention, such as from the use of tissue engineered constructs. Recent studies have increasingly used
Available online 3 March 2014
co-cultures in tissue engineering applications as these systems better model the natural tissues, both
physically and biologically. This review aims to identify the challenges of using co-culture systems and to
Keywords:
highlight different approaches with respect to the use of biomaterials in the use of such systems. The
Co-culture
application of co-culture systems to stimulate a desired biological response and examples of studies
Biomaterials
Tissue engineering
within particular tissue engineering disciplines are summarized. A description of different analytical co-
Cellular interactions culture systems is also discussed and the role of biomaterials in the future of co-culture research are
Stem cells elaborated on. Understanding the complex cellecell and cellebiomaterial interactions involved in co-
Monocytes culture systems will ultimately lead the field towards biomaterial concepts and designs with specific
biochemical, electrical, and mechanical characteristics that are tailored towards the needs of distinct co-
culture systems.
Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction physical interactions [8]. Due to various physiological events such

In the physiological environment, natural tissues most often
consist of multi-cellular systems of two or more types of cells which
interact with one another to facilitate viability, proliferation [1,2], as
well as generating growth factors and proteins [3,4], promoting
differentiation [5], and enabling other key biological tasks. For
example, the multi-cellular system of endothelial cells (ECs),
vascular smooth muscle cells (VSMCs), and fibroblasts make up the
blood vessels [6], and the organized structure of epithelial cells,
gingival fibroblasts, periodontal ligament cells, ECs, and osteoblasts
generate periodontal tissues [7]. In arteries, cellular cross-talk be-
tween ECs and VSMCs regulates the proliferation of the VSMCs [2],
preventing the hyperproliferative VSMC phenotypic state that is
characteristic of pathological conditions. In periodontal tissues,
gingival fibroblasts regulate the migration of epithelial cells via
* Corresponding author. Department of Biomaterials, Faculty of Dentistry, Uni-
versity of Toronto, 124 Edward Street, Room 464D, Toronto, Ontario, Canada M5G
1G6. Tel.: þ1 416 979 4903x4341; fax: þ1 416 979 4760.
E-mail addresses: paul.santerre@utoronto.ca, paul.santerre@dentistry.utoronto.
ca (J.P. Santerre).
1
These authors contributed equally to this work.
as disease or injury, some natural tissues, like neural tissues, may
not regenerate themselves or, in some cases, the body may attempt
to repair damage but the resulting tissues do not have the same
properties and function of the original tissue (e.g. the generation of
the fibrotic tissues in the myocardium after an infarction [9]). In an
attempt to regenerate these damaged tissues and organs, tissue
engineering approaches have attracted great interest.
For a long time, many biomaterial studies have focused on single
cell studies with such constructs. However, recent directions in the
field have increasingly used co-cultures in tissue engineering as
these systems better model the natural tissues, physically and
biologically, through interactions between different cell types.
Despite the increasing interest in using co-culture systems for tis-
sue engineering purposes, challenges have been identified from
these co-culture studies (Table 1), which place constraints on the
design of the experiment, and ultimately influence the effective
analysis of the experimental results. In particular, co-culture sys-
tems involve multiple interactions (Fig. 1) that can be difficult to
distinguish from one another without the use of proper experi-
mental design, which can lead to the misinterpretation of results.
For example, a significant barrier to the effective analysis of co-
culture systems is being able to distinguish the contribution of the

http://dx.doi.org/10.1016/j.biomaterials.2014.02.023
0142-9612/Ó 2014 Elsevier Ltd. All rights reserved.
4466 K.G. Battiston et al. / Biomaterials 35 (2014) 4465e4476

Table 1
Challenges associated with co-culture experiments.

Issue Challenge References for relevant studies

Medium optimization If two cell types have different optimized growth media, for co-culture experiments [5,23,27,29,35,55,72,78,90,113]
which growth medium (or combination of growth media) should be used to
optimize growth and cell phenotype? Should growth supplements still be
added since cellecell contact between cell types may already promote
specific responses?
Cell type/origin The anatomical origin of the cells (ex. microvascular ECs, HUVECs, HAMECs, etc.) [1,3,4,9,23,25,27,31,34,46,57,
may influence the outcome of the experiment, and the appropriate cell type 59,71,87,92,98]
should be chosen for the organ system that the co-culture experiment
needs to address.
Cell ratio What cell ratio will lead to optimal experimental outcomes? [23,30,31,39,72,78,90,92,93,160]
Cell number If experiments are continued from a monoculture, does one maintain the [23,67,94,99,121,140]
same cell number used in the monoculture study for each cell type? Or is
an optimized cell ratio required? Should the total cell population vary?
Seeding protocol Should cells be seeded simultaneously or sequentially? If sequentially, which [2,14,20,28,32,36,38,55,67,70,
cell type should be seeded first? 78,88,108,138,139,160,161]
Static or dynamic Should additional mechanical stimulus be introduced in the culture? [75,81,96,108,129,162]
3D vs. 2D Will the cells in the co-culture system behave differently in a 2D vs. 3D setting? [30,54,77,82,107,108,128,155]
Location of cells within scaffold Is spatial patterning necessary? [28,36,75,77,89,100,101,104,106,
108,115,120e123,133,135e138,
140,149,161]
Purpose of the experiment One must consider if it is necessary to precisely mimic the physiological system [125e129,131,132,163]
in order to maximize specific experimental outcomes.
Optimization conditions When optimizing experimental conditions, it is important to choose the correct [25,33,36,38,56,67,85,93,99,104,
analysis parameters for the optimization (i.e. maximized cell growth, enhanced 105,114,124,134,136,139,
cellular differentiation, maximized phenotypic marker expression, maximized 140,147]
matrix synthesis, etc.).
Distinguishing cell types from one another Appropriate and selective phenotypic markers must be chosen to distinguish [85,93]
between the cell types.
Contribution of different cell types to If both cell types express the same phenotypic markers, co-staining for multiple [10,11]
proliferation or phenotypic markers may be a possible solution. Careful selection of stains (e.g. fluorescent labels)
marker expression and sample preparation are important. Vital dyes can be used to label different cell
types prior to seeding onto biomaterials and imaging or flow cytometry can be used
to determine spatial distribution of each cell as well as the increase in cell number
of each cell type individually.
Distinguishing paracrine vs. cellecell In typical co-culture systems, the effects mediated by cellecell contact vs. paracrine [1,21,25,31,35,69,71,72,105,
contact mediated effects signaling cannot easily be separated. Strategic experimental design is required to 116,121,122,135,136,146,
disentangle the two from one another to determine mechanistically what is 148,155]
happening within the co-culture system to properly inform future research.

different cell types to the measured responses. More specifically,
typical assays used to assess proliferation or metabolic activity may
only measure the response of the system as a whole, but will not
provide insight into which cell type in the system is proliferating and
at what rate. There are a number of strategies that can be employed
to differentiate between cell types to address this issue. Perhaps one
of the most powerful involves the use of vital dyes [10,11], whereby
labeling each cell type allows the fate of each cell type to be tracked.
It may also be necessary to perform immunostaining for cell type-
specific markers to determine if the initial seeding ratio has been
maintained throughout the length of the experiment.
Another factor that can be lost during data interpretation is the
relative contributions of cellecell interactions versus cellebioma-
terial interactions to the observed responses. This issue can
partially be addressed through the use of control monoculture
conditions, wherein the cell types present in the co-culture system
are cultured in parallel by themselves but otherwise under identical
conditions (medium type, biomechanical stimulation, culture
substrate, etc.) to the co-culture. This will aid the experimenter in
determining if the effects of the co-culture are altering the effects of
the cells compared to how they are stimulated by the biomaterial
substrate alone. However, this type of experimental design will not
determine if cellecell communication or the stimulus imparted by
the biomaterial is the dominant factor in the culture system. While
the biomaterial substrate is always likely to play an important role,
conducting the same co-culture on different biomaterials will help
determine if the stimulus provided by the biomaterial has potential
importance in the outcome of the co-culture system, in addition to
the effects caused by the interaction of the two cell types.
Synthetic and natural biomaterials play a critical role in tissue
engineering as they can modulate cell response via different ma-
terial properties such as surface chemistry (i.e. presence of func-
tional groups, hydrophobicity, ionic and hydrogen bonding, etc.),
topography, roughness, compliance, porosity, isotropy, etc [6]. Cell-
biomaterial interactions also affect cellecell interactions in co-
culture systems, in that all cell types in the co-culture environ-
ment will behave uniquely upon interaction with different bio-
materials, which may affect how one cell type interacts with the
other. This can be advantageous in developing new approaches for
promoting tissue regeneration.
This review aims to highlight the different approaches to co-
culture systems with biomaterials, while identifying key studies
that have been able to overcome some of the challenges associated
with such systems (Table 1). Specifically, the use of co-culture
systems to stimulate a desired biological response and examples
of studies within particular tissue engineering disciplines will be
summarized and key studies highlighted. Furthermore, the use of
different analytical systems to probe cellecell interactions in co-
cultures, and their relative advantages and disadvantages will be
discussed. Lastly, this review will report on the future promise of
strategically directed biomaterial development towards enhancing
biological outcomes in co-culture systems and the use of bioma-
terial concepts to address some of the challenges inherent to this
field of work.
 K.G. Battiston et al. / Biomaterials 35 (2014) 4465e4476
Fig. 1. Different interactions that occur in co-culture systems. Cells have multiple ways of interacting with one another. It can be difficult in co-culture systems to distinguish how
the cells are communicating, and whether the effects observed are primarily due to cellecell communication, cellebiomaterial interactions, celleprotein interactions, or autocrine
effects, such as when a stimulatory molecule for one cell type can be produced by both cells.
2. Promotion of biological responses
Co-culture systems can be used with the intent of using one cell
type to provide a desired stimulus to a second cell type. This
strategy takes advantage of the naturally occurring cross-talk be-
tween cell types, either through soluble factors or direct cellecell
contact, which occurs in healthy tissues [12], regeneration [13],
wound healing [14], and during developmental processes [15]. This
can often be a less cost-intensive strategy than dosing specific
growth factors into culture medium to provide a similar effect.
2.1. Promotion of vasculogenesis and angiogenesis
The long-term success of tissue engineering strategies both
in vitro and following implantation relies significantly on the ability
of cells to receive an adequate supply of nutrients, particularly for
large and deep tissues that are susceptible to diffusive limitations
[16]. Most tissues are highly vascularized (e.g. bone [17]) and thus
need to be engineered with a vascular network in place to support
nutrient transport and to ease the connection with the host vascu-
lature upon implantation. Seeding endothelial cells (ECs) on
biomaterial scaffolds for this purpose has not been an effective
strategy due to the inability of ECs to survive and proliferate for long
culture times, as well as their inability, in monoculture, to self-
assemble and form tube-like structures [18]. However, a strategy
that has proven effective involves the co-culture of ECs with
different cell types, which provides stimulation through the release
of soluble factors (e.g. vascular endothelial growth factor (VEGF) and
platelet-derived growth factor (PDGF)), resulting in the self-
assembly of ECs into capillary-like structures in vitro [19,20]. Pri-
marily, ECs have been successfully co-cultured with fibroblasts [3]
and osteoblasts [21] to support cell proliferation and capillary for-
mation, though stem cells have shown promising results as well [22].
Exogenous supplementation of growth factors to promote EC
function in vitro often involves VEGF, basic fibroblast growth factor
(bFGF), epithelial growth factor (EGF), insulin-like growth factor-1
(IGF-1), or some combination thereof [23]. The effects of fibro-
blasts and osteoblasts supporting angiogenesis in co-culture sys-
tems with ECs have primarily been linked to paracrine signaling
mechanisms [24]. Co-cultures of fibroblasts with ECs have been
shown to stimulate VEGF release by fibroblasts, upregulate VEGF
receptor 2 on ECs, and support endothelial nitric oxide synthase
and nitric oxide production from ECs [24]. Similarly, short-range
4467
paracrine signals have been shown to be important for maintain-
ing EC phenotype in a hepatocellular model involving hepatocytes
and fibroblasts [25], while other studies have also shown similar
effects [26,27]. The hepatocellular model illustrates the use of a
unique system to allow for the deconvolution of effects mediated
by cellecell contact vs. soluble signals [28]. This involves seeding
cells on separate silicone microchips which can then be brought
into contact or kept at a distance (‘gap culture’, 80 mm separation) in
order to assess any effects mediated by cellecell contact or through
paracrine mechanisms. In other systems, however, the effects of
different cell types on ECs have been shown to be dependent on
cellecell contact rather than through paracrine signaling [29e31].
It should also be noted that paracrine mechanisms have been
shown to be responsible for enhanced angiogenesis [19,25], while
reduced angiogenesis and lack of responsiveness to angiogenic
factors have been linked to cellecell contact [29e31]. Co-culture
systems designed for promoting angiogenesis should therefore
look to provide an environment with appropriate cell distribution
and density to allow for the soluble signal-mediated pro-angio-
genic effects of co-culture, without the suppressive effects induced
by cellecell contact. This need for fine control of the cellecell ratios
in co-cultures provides an opportunity to capitalize on the field’s
ability to conceive synthetic and natural biomaterial strategies that
can perturb cell proliferation, migration, and distribution by vary-
ing the chemical, electrical, and mechanical properties, as well as
the interconnectivity, of biomaterial constructs.
An example of in vitro vascularization providing benefits post-
implantation was demonstrated by Guillemette et al., where a
fibroblast-cell sheet was seeded with ECs, resulting in the forma-
tion of capillary-like structures after 7-days [32]. When the cell
sheet was then rolled up to a form a tissue-engineered vascular
construct, the result was a vascularized blood vessel that had
improved blood circulation and enhanced integration with the
surrounding environment following implantation compared to
non-pre-seeded constructs [32]. The success of EC co-culture sys-
tems, primarily with fibroblasts or osteoblasts, is an example of
how co-culture systems can be used to provide stimuli to one cell
type through culture with a second cell type exhibiting a pheno-
type conducive to supporting a particular biological outcome.
While this section has focused specifically on the role of cells
supporting angiogenesis when co-cultured with ECs, it should be
noted that ECs in these systems are also playing important roles in
regulating the phenotypic state of the other cells [33e36].
4468 K.G. Battiston et al. / Biomaterials 35 (2014) 4465e4476
2.2. Monocytes and co-culture systems
Monocytes are a key cellular component involved in the wound
healing response to biomaterials following implantation [37].
Depending on their activation state, monocytes and their derived
macrophages will release a spectrum of pro-/anti-inflammatory
and/or pro-/anti-wound healing growth factors and cytokines that
will determine, to a significant extent, the ability of the material to
support tissue integration and regeneration. As a result, in vitro co-
cultures containing monocytes can be used as a strategy to promote
other cell types to adopt a phenotypic state that is conducive to
supporting tissue formation and regeneration, as well as cell
growth or other properties depending on the application [38,39]. In
particular, there has been a recent focus in the biomaterials liter-
ature leading to an understanding that monocyte/macrophage
dysfunction with respect to monocyte/macrophage activation in
healthy tissue is associated with adverse outcomes, such as the case
of obesity where macrophages in adipose tissue have been polar-
ized to an M1 (pro-inflammatory) phenotype, compared to lean
individuals where macrophages in adipose tissue adopt a pre-
dominantly M2 (pro-wound healing, regulatory) phenotype [40].
Co-culture systems with monocytes/macrophages that are induced
into a pro-wound healing, anti-inflammatory state by biomaterials
have potential as strategies for tissue regeneration [41].
Surface properties, such as topography and chemical function-
ality, have been shown to be important mediators of the monocytic
response to synthetic biomaterials [42e45]. The diverse phenotypic
range of monocytes/macrophages and the ability of biomaterials
with specific properties to elicit distinct activation states provides a
unique opportunity to develop biomaterial strategies to promote a
monocyte/macrophage activation that can support a desired effect
in different co-culture systems. It has recently been shown that
monocytes can promote the migration and growth of VSMCs when
they are co-cultured on a degradable polyurethane [38]. Further-
more, monocytes seeded on an acrylamide-based hydrogel that
were highly expressing CD163, an M2 marker of activation, were
shown to support human umbilical vein EC (HUVEC) proliferation
through the release of VEGF, without releasing pro-inflammatory
cytokines, compared to control monoculture conditions [46].
Enhanced cell growth can accelerate the process of tissue formation
in vitro, which is essential before cell-seeded biomaterials are ready
to be implanted. Furthermore, monocytes can non-invasively be
isolated from a patient’s peripheral blood, making them an easily
accessible, autologous source of stimulatory cytokines and growth
factors. In comparison, the addition of exogenous growth factors
such as VEGF, PDGF-BB, or EGF can quickly become expensive,
particularly with long culture periods, which is further complicated
by the possibility of endotoxin contamination if the proteins are
obtained from a recombinant source [47].
Culturing monocytes and other cells of the inflammatory system
on biomaterials ex vivo is useful for providing initial insight into
how a new biomaterial is going to be seen by elements of the body’s
immune system, and whether an adverse foreign body reaction is
to be expected or if beneficial wound healing and tissue regener-
ation could be anticipated. However, there are several issues
associated with the use of immune cells that can compromise the
integrity of the collected data. A large amount of studies reporting
the use of immune cells in vitro rely on the use of fetal bovine serum
or other xenogeneic protein sources to culture their cells. While
traditional in vitro culture systems are already limited in terms of
their predictive capacity for how biomaterialecell interactions will
occur in vivo, the use of xenogeneic media with immune cells in
particular can give skewed results. It has recently been shown that
culturing cells on the same biomaterial substrate, but in the pres-
ence of human serum vs. xenogeneic serum, can result in
differences of up to two orders of magnitude in the release of
specific proteins [48]. Proteins in human sera in some cases have
been shown to be more bioactive towards human cells than the
bovine source of the same proteins [49].
In addition to the use of immune cells ex vivo to investigate celle
biomaterial interactions, immune cell cultures can also be used to
understand cellular cross-talk between immune cells and other cell
types in different co-culture systems. The use of immune cells for
this purpose, however, should be viewed with some skepticism, as
the previously mentioned used of xenogeneic media can produce
misleading results. Furthermore, the outcome of poly-
morphonuclear leukocyte-monocyte co-cultures has been shown
to have a biomaterial dependence [50], such that any insight pro-
vided by the in vitro culture system is relative to the culture sub-
strate that was used, and does not necessarily provide insight with
regards to the cellular cross-talk that will occur in other environ-
ments. Even the use of TCPS, the most common culture substrate
for evaluating cellecell interactions, is known to vary depending on
the manufacturer, with numerous reports demonstrating varied
cell responses, including its use with monocytes, to TCPS from
different manufacturers [51e53]. Care must thus be taken when
interpreting data from reports involving the use of immune cells on
different biomaterials as well as in the presence of different media
types, while also giving thought to the source of the monocytes and
the distribution of their phenotypes.
2.3. Co-culture to induce stem cell growth and differentiation
Stem cell growth in vitro, in order to maintain the undifferen-
tiated state of the stem cell, requires the presence of certain growth
factors, cytokines, and nutrients. This is often accomplished with
the use of feeder cells, typically fibroblasts, in a process where the
feeder cell layer is used as a substrate to grow the stem cells. The
effects of the feeder layer on stem cell survival and expansion can
be mediated through cellecell contact, feeder layer-assembled
extracellular matrix (ECM), or various cytokines and growth fac-
tors [54]. In contrast to this more traditional use of co-culture to
maintain an undifferentiated stem cell state, co-culture systems can
also be used with the goal of inducing stem cell differentiation.
Several examples include the co-culture of osteoblasts with
mesenchymal stem cells to promote osteoblastic differentiation
[55], osteoblasts enhancing hematopoietic progenitor cell prolif-
eration and function [56], ECs regulating cardiomyocyte develop-
ment from embryonic stem cells (ESCs) [57], and the differentiation
of placenta-derived multipotent cells to neuronal and glial cells
under co-culture with rat brain cells [58].
Stem cell differentiation or self-renewal can be managed by
using the appropriate co-culture system. This has recently been
illustrated by Sneddon et al., where it was shown that the prolif-
eration and self-renewal of ESC-derived progenitors, without dif-
ferentiation, could be promoted by co-culture with organ-matched
mesenchyme [59]. This study also illustrates that not only is the cell
type being co-cultured an important, but also that the source of this
cell can have important implications. To promote differentiation,
ESCs have been co-cultured with ECs to promote the formation of
pancreatic insulin producing cells [60]. Co-cultures of ESCs with
primary bone-derived cells on PLGA/HA composite scaffolds,
however, has been shown to be efficient for osteogenic differenti-
ation [61].
Within these co-culture systems there are two important factors
that can be used to favor a particular differentiated state, namely
the cell type being used for the co-culture, as well as the bioma-
terial substrate upon which the culture will be performed, where
the different mechanical, electrical, chemical, and morphological
properties of the substrate can play important roles. To optimize
 K.G. Battiston et al. / Biomaterials 35 (2014) 4465e4476
the different aspects of the co-culture system, including the cell
type and designed properties of the biomaterial, the in vivo differ-
entiation conditions under which stem cell fate is specified should
be taken into consideration. For example, substrate elasticity has
been shown to favor ESC differentiation to different germ layers
[62]. In a study by Zoldan et al., 3D scaffolds were engineered to
possess different stiffness thresholds which are similar to the
different forces that cells would be exposed to during the process of
gastrulation, where it is known that mechanical forces are critical
to embryogenesis [62,63].
As discussed in Section 2.2, monocytes/macrophages can be a
source of growth factors and cytokines, with the exact make-up of
their secretome dependent on their activation state [64]. These
cells have recently been shown to provide directed differentiation
of stem and progenitor cells dependent on their culture environ-
ment and their particular phenotype. Harnessing the power of
secreted products from a particular phenotype has also been
exploited for MSCs [65]. When using collagen-based materials for
cell entrapment, macrophages were shown to attenuate MSC dif-
ferentiation to chondrocytes, but enhance osteoblast differentia-
tion [66]. The ability of macrophages to direct differentiation was
shown to be material-dependent, with cultures contained within a
gelatin/PEG-based matrix promoting adipocyte differentiation [66].
Macrophage effects on MSCs has also shown a polarization de-
pendency, with M1 macrophages inhibiting growth and M2 mac-
rophages promoting growth [67]. In other studies, MSCs have been
shown to be important mediators of macrophage polarization [68e
70], such as promoting the regulatory, or M2, state [68,69]. In
addition to supporting adipocyte, osteoblast, and chondrocyte
differentiation, macrophage co-culture has also been shown to
support MSC differentiation to SMCs when the macrophages have
been activated with IL-1b [71]. Macrophage effects on MSC differ-
entiation have been shown to be mediated by both cellecell contact
[72] and the release of soluble factors in a paracrine manner [71,72],
where effects in this particular study were determined to be cell
contact dependent based on the use of a transwell system with a
0.4 mm semipermeable membrane [72].
3. Rebuilding of natural tissues
Most co-culture systems are developed to repair, regenerate, or
mimic the natural tissues. These systems often involve two or more
types of cells cultured in a biomaterial but vary in terms of the
sequence in which the cells are seeded, the spatial distribution of
different cell types, and the structure of the biomaterials. In this
section, examples of co-culture systems used with biomaterials in
the regeneration of various types of tissues will be discussed.
3.1. Cardiovascular tissue regeneration
3.1.1. Vascular tissues
A good number of co-culture systems have been established for
vascular tissue regeneration with the goal of replacing damaged
vessels [73] and reconstructing the vessel environment [2,4,74e76].
Co-cultures of vascular ECs with VSMCs have exhibited a higher
survival rate for the vascular ECs [4,74e77] and promoted the
proliferation of VSMCs [2,77], indicating their potential to generate
vessel-like structures. In most studies, ECs and VSMCs were seeded
in a mixture, and the cells were allowed to rearrange themselves to
form two distinct layers of ECs and VSMCs via cellular crosstalk
[77]. However, ECs may initially migrate into 3D scaffolds and,
instead of forming a monolayer, they may establish a capillary-like
network [78], which results in a different tissue structure from the
natural blood vessel. As such, sequential seeding was performed in
which the VSMCs were seeded first to form a basement layer,
4469
followed by the seeding of ECs to generate a tissue-engineered
vascular graft (TEVG) [4]. Scaffold design plays an important role
in the generation of a TEVG. For example, the pore interconnectivity
and the porosity of the scaffolds allow interactions between cell
types [38,79] and uniform cell distribution [4]. The structure of the
scaffold (i.e. layered and porous [75,80,81] is also important for
mimicking the natural blood vessel.
Mechanical stimuli such as perfusion and cyclic strain have also
been added into co-culture systems in order to promote cell pro-
liferation [75,76,80] and to modulate the phenotype of VSMCs
[75,76]. Work by Williams and Wick used a perfusion bioreactor
and co-cultures with ECs in order to promote cell proliferation,
uniform cell distribution, and induce a more contractile phenotype
in VSMCs [76]. The use of pulsatile flow with a silk fibroin scaffold
was also shown to allow for the formation of an EC layer on top of
the VSMCs, while promoting the proliferation and alignment of
VSMCs, retaining the contractile phenotype of the VSMCs, as well as
enhancing extracellular matrix production [75]. In order to further
stimulate cell responses, additional cell types such as monocytes
have also been introduced into co-cultures with ECs and VSMCs to
modulate the cells’ phenotypes. Monocytes in the wound healing
state have been shown to induce a less contractile phenotype in
VSMCs [79]. Also, polystyrene-activated monocytes and neutro-
phils (i.e. in a pro-inflammatory state) have been reported to
downregulate EC proliferation [4], indicating that a less pro-
inflammatory monocyte phenotype may be beneficial in EC-
VSMC co-culture systems.
3.1.2. Cardiac tissues
After myocardial infarction, cardiac tissues become damaged
but they have difficulty to be regenerated by the body as the car-
diomyocytes are terminally differentiated. Instead, fibrosis occurs
and results in the formation of a fibrotic scar and organ dysfunction.
Cardiac tissue engineering has been focused on the use of car-
diomyocytes to generate a cardiac patch that can be used to replace
the damaged tissue; however, these cells could not remain viable in
long-term monoculture and sometimes could not contract syn-
chronously [9]. It has been shown that co-culturing cardiomyocytes
with cardiac fibroblasts on two-dimensional tissue culture plastic
and in three-dimensional collagen matrices promoted synchro-
nized contraction in the cardiomyocytes with the presence of
intercalated discs [82]. Recently, a similar co-culture model was
built on electrospun chitosan nanofiber scaffolds. In this study, the
cardiomyocytes exhibited polarized morphology and their lifespan
was prolonged by the presence of fibroblasts [9]. The co-culture of
engineered cardiac progenitor cells with neonatal myocytes has
been shown to promote the commitment of the myocyte lineage in
the cardiac progenitor cells and the resulting construct had elec-
trophysiological properties that are similar to mature cardiac tis-
sues [83].
3.2. Bone and cartilage tissue regeneration
3.2.1. Bone tissues
Bone is a calcified matrix consisting mainly of osteoblasts
residing in a highly vascularized network. In bone regeneration,
osteoblasts are often co-cultured with ECs to induce capillary for-
mation [19,21]. ECs, however, can similarly affect osteoblast
phenotype and proliferation. Osteoblast-EC co-cultures have been
reported to upregulate the expression of osteogenic markers (e.g.
ALP) and to support osteoblastic proliferation of osteoprogenitors
[33,84]. Similar co-cultures on silk fibroin scaffolds consisting of
porous hydroxyapatite, calcium phosphate and nickel-titanium
showed that osteoblast-EC crosstalk prolonged the lifespan of ECs
and promoted the formation of microcapillary structures, while ECs
4470 K.G. Battiston et al. / Biomaterials 35 (2014) 4465e4476
in the monoculture began to die after one week of culture [85]. In
addition, fibroblasts have been reported to support osteoblast dif-
ferentiation and ectopic bone formation [86] and to support lacuna
structure by co-culture with chondrocytes on biodegradable PLGA
(poly-lactic-glycolic acid) scaffolds [87].
While co-cultures of osteoblasts with other cell types have
shown promising results in bone regeneration, the osteochondral
interface (i.e. cell interaction between the bone and cartilage) is not
well established. Studies of osteoblast-chondrocyte co-cultures are
currently being assessed to regenerate the osteochondral interface
[88]. It has been shown that by co-culturing osteoblasts with
chondrocytes, ALP activity was maintained in osteoblasts, but
glycosaminoglycan (GAG) content was decreased, along with
decreased mineralization in general. The mechanisms that resulted
in the aforementioned cell behavior remain to be investigated. In
addition, the spatial distribution of the two cell types is important
in regulating cell behavior such as maintaining the chondrogenic
phenotype in chondrocytes on one side of a bilayered agarose-
PDMS scaffold while promoting ossification on the other side [89].
Various challenges in co-culture systems for bone regeneration
should be addressed during experimental design and these include,
but are not limited to, the sequence of cell seeding as well as the cell
seeding ratio (i.e. osteoblast:EC). For regenerating bone with a
microcapillary network, osteoblasts and ECs are often seeded
together [19,21,33,85], but for generating an osteochondral inter-
face, sequential seeding should be considered [88]. Various seeding
ratios have been explored in these co-culture systems. A recent
study has shown that the highest mineralization was observed for a
50:50 ratio of osteoprogenitor cells and ECs [90]. The choice of
culture medium is also important in the co-cultures for bone
regeneration as the medium should (i) promote bone mineraliza-
tion and (ii) EC proliferation, along with subsequent capillary
formation.
3.2.2. Cartilage tissues
Similar to the cardiac tissues, articular cartilage tissue has
limited regenerative capacity and the phenotype of chondrocytes, a
major cell type in articular cartilage, is difficult to maintain in a
tissue-engineered construct for long-term culture [91]. In cartilage
tissue engineering, chondrocytes have been co-cultured with
various cell types for their phenotypic maintenance and to rebuild
cartilage-like tissue. For example, chondrocytes embedded in
agarose gel have been co-cultured with human MSCs in separate
bilayers (5  3 mm) such that the MSCs were physically separated
from the chondrocytes. It was found that the MSCs provided
paracrine signals to the chondrocytes, which resulted in chondro-
genesis and promotion of type II collagen, a phenotypic marker of
chondrocytes, with no mineralization in the chondrocyte layers
[89]. While this study used a 1:1 cell ratio, a similar study by
Meretoja et al. showed that a 1:3 ratio of chondrocytes:MSCs in
porous polycaprolactone scaffolds (8 mm in diameter) had
enhanced cartilage-like matrix production [92]. There is no
consensus on the optimized ratio that enhances chondrocyte pro-
liferation and cartilaginous ECM production. Chondrocyte-MSC co-
culture has also regulated the hypertrophic behavior in MSCs, thus
reducing mineralization in the engineered cartilage [93]. Other
types of stem cells such as adipose-derived stromal cells have been
co-cultured with chondrocytes in polyglycolic acid/polylactic acid
scaffolds. After eight weeks, the resulting tissue-engineered con-
structs were cartilage-like with rich type II collagen and GAG
contents [94]. Furthermore, chondrocytes from different parts of
the body have also been used to regenerate articular cartilage.
Costal chondrocytes were harvested and co-cultured with dental
pulp stem cells at a ratio of 5:5 or 3:7 to enhance chondrogenesis. A
ratio of 5:5 was chosen to minimize mineralization; however
exogenous fibroblast growth factor 9 was still required to inhibit
mineralization in the engineered cartilage [95]. In addition, a me-
chanical stimulus (intermittent hydrostatic pressure) has been
introduced to the co-culture of chondrocytes and MSCs in alginate
beads, and at higher magnitudes, the intermittent hydrostatic
pressure has been shown to enhance the proliferation and differ-
entiation of MSCs into chondrocytes [96].
3.3. Neural tissue regeneration
Co-culture has also proven to be an important strategy towards
achieving neural regeneration. PLGA multiple-channel conduits
have been used to co-culture Schwann cells and MSCs, which pro-
mote MSCs to differentiate into neuron-like cells as well as forma-
tion of synapse-like structures [97]. Schwann cells have also been
co-cultured with adipose-derived stem cells (ASCs) in a transwell
system (Millicell) to promote Schwann cell-like differentiation [98].
Schwann cells co-cultured with neurons in a hydrogel network of
collagen and hyaluronic acid also showed improved neurite growth
[99]. Patterned biomaterials such as multi-channel-patterned PLG
(Poly-Lactic-Glycolic) have been used to direct neurite extension in
co-cultures of primary neurons and accessory cells [100]. Other co-
culture systems for neural regeneration have been developed such
as those used for encouraging the formation of neuromuscular
synapses in vitro [101,102]. Intrafusal muscle fibers were co-cultured
with type Ia sensory neurons on a surface-patterned PEG-silane
substrate as well as MEMs device to study the conversion of me-
chanical information from the intrafusal fibers to electrical action
potential in neuromuscular synapses. The resulting cell morphology
and calcium ion response in the co-culture system were similar to
those observed in physiological synapses [101]. As neurons can be
electrically stimulated, biomaterials such as hydrogels can be
positively charged to stimulate neurite outgrowth [103]. It should
also be noted that different spacing widths between these micro-
channels/patterns can affect electrophysiological interactions be-
tween neurons and other cell types.
3.4. Other tissue regeneration
3.4.1. Periodontal tissues
Periodontal tissue engineering involves the regeneration of the
gingival tissue, the periodontal ligament and the alveolar bone [7].
In order to reconstruct the damaged gingiva, gingival fibroblasts
have been co-cultured with gingival keratinocytes on poly-
dimethylsiloxane (PDMS) pillars to promote epithelial cell
morphogenesis via cellecell contact between the two cell types
[8,104]. Co-cultures have also been used to promote osteogenesis
and address the bone loss caused in destructive periodontal dis-
eases. For example, periodontal ligament stem cells (PDLSCs) were
co-cultured with human umbilical vein ECs in a transwell setting
with polycarbonate membranes to induce osteogenesis in PDLSCs
[105]. Furthermore, in order to prevent keratinocytes or gingival
fibroblasts from migrating into the periodontal ligament and over-
populating the periodontal defect, a method called guided tissue
regeneration was used such that the biomaterial scaffold, typically
ePTFE (expanded polytratra-fluoro-ethylene) or PLGA, provided a
barrier between the periodontal ligament cells and the gingival
keratinocytes and fibroblasts [106].
3.4.2. Liver tissues
The regeneration of liver tissues requires careful spatial distri-
bution of different cell types and the use of a 3D structure is
important for priming specific cell behavior as well as establishing
cellular crosstalk between the cells of interest. Studies have been
carried out to mimic liver tissue by co-culturing rat hepatocytes
 K.G. Battiston et al. / Biomaterials 35 (2014) 4465e4476
with rat stellate cells on a PLGA substrate [107]. Viable 3D spheroids
were formed at a faster rate in co-culture as compared to hepato-
cyte monoculture, and higher expression of hepatocyte growth
factor mRNA was observed in the co-culture, which indicates the
potential of this co-culture to regenerate liver tissue. Hepatocytes
have also been co-cultured with ECs in PDMS honeycomb micro-
wells to form endothelialized aggregates, which were then mixed
with biodegradable poly-L-lactide fibers such that the aggregates
were spaced out [108]. The hepatocyte-EC interaction in this spatial
orientation and the influence of the biomaterial had increased al-
bumin production and the aggregates remained spherical.
3.4.3. Skin tissues
Co-culture systems for skin regeneration were extensively
developed in the 90’s and they predominately involved co-cultures
of fibroblasts and keratinocytes. Early co-cultures of keratinocytes
and dermal fibroblasts on three-dimensional constructs showed
success in reconstructing the epidermis [109e112]. For example,
after 5 weeks of in vitro culture on 3D nylon mesh, the co-culture of
human dermal fibroblasts with human keratinocytes showed
epidermal differentiation; and the basal lamina, anchoring zone,
and dermis were distinctly present with corresponding markers
[109,110]. This type of co-culture system has also been used to
reconstruct the stratified epidermis for cytotoxicity testing
[111,112]. Fibroblast-keratinocyte co-culture systems have been
shown to regulate collagen synthesis [113], induce myofibroblast
differentiation [114], and to induce fibroblast migration [115].
In addition to fibroblast-keratinocyte co-culture, dermal
fibroblast-EC and fibroblast-stem cell co-culture systems have been
used [116e118]. Studies on dermal fibroblast-EC co-culture systems
could be dated as early as the 1990’s. In early studies of angio-
genesis, dermal fibroblasts were co-cultured with human umbilical
vein ECs in vitro on 2D culture flasks to demonstrate the potential of
this co-culture system for capillary formation in skin regeneration
[117]. Vascularization was also observed in dermal fibroblast-EC co-
culture systems in 3D collagen scaffolds, and the resulting construct
showed integration with the host vasculature 4 days after im-
plantation [118].
Different biomaterials such as fibrin gels [115] and collagen
matrices [119] have been used in co-culture systems for skin
regeneration. The cell behavior in co-culture systems is dependent
on the biomaterial used. For example, stratified layers of kerati-
nocytes were formed on the surface of both fibrin gels and collagen
matrices, along with fibroblast migration into the core of the
scaffolds. However, more fibroblast migration was observed in the
collagen matrices in a shorter time frame than in the fibrin gels. In
addition, the use of different media may enhance or retard fibro-
blast migration [115].
4. Analytical systems for studying cell response in co-culture
In addition to the use of co-culture systems for tissue regener-
ation and stimulation of biological responses, these systems can
also be applied to study the fundamental aspects of cellecell
interaction in specific tissues, developmental biology, as well as to
examine cell responses to specific stimuli (tissue modeling). In this
section, the use of micropatterning and microfluidic systems with
co-cultures used for tissue modeling will be discussed.
4.1. Micropatterning and microfluidic systems e vascular
microenvironment
Micropatterning is often used to study the interactions between
two cell types in a co-culture. For example, gelatin films were coated
with bands of a cell-resistant polyelectrolyte coating and ECs were
4471
seeded in the channels where the coating was not present. The
entire surface was then covered with chitosan and a layer of fibro-
blasts were seeded on top to investigate fibroblast-induced capillary
formation in a well-oriented space [120]. Micropatterning can also
be used to control the area in which cellecell interaction can occur as
well as the cell populations that are allowed to interact with one
another. Using micropatterning, fibroblasts were confined at the
periphery of hepatocyte islands and by controlling the area of pro-
tein coating, the distance between these islands and fibroblast
density changed. These arrangements were useful for investigating
fibroblastehepatocyte interactions with controllable parameters
[121]. Similarly, in a study by Lamponi et al., ECs and fibroblasts were
seeded in rectangles of various areas that were micropatterned on
silanized glass. By varying the size of the areas, the morphology of
fibroblasts changed and the number of fibroblasts allowed to
interact with ECs was limited by the area of the rectangle [122].
Microfluidic systems can create a physiologically-relevant
microenvironment that models the vasculature while reducing
size and cost [123]. At this micro-scale, the spatial and temporal
aspect of the cell environment such as cell distribution and diffu-
sion rate can be better controlled to mimic three-dimensional tis-
sue architecture and study cell behavior [124]. Microfluidic systems
have often been used to study paracrine effects induced in the co-
culture of ECs and various cell types on hydrogels [125,126]. For
example, stromal cells embedded in fibrin gel channels promoted
capillary formation from the ECs cultured in the adjacent channel
[126]. Microfluidic devices have also been used to study how
different growth factors patterned on the microfluidic platform can
affect ECs in co-cultures. Extracellular matrix proteins have also
been used to form microstructured platforms to spatially organize
breast cancer cells adjacent to ECs for investigating tumor angio-
genesis [127].
4.2. Tissue modeling
Co-cultures have also been used in tissue modeling for (i) drug
screening and (ii) studying various diseases such as cancer
metastasis and respiratory diseases. In terms of drug screening, co-
cultures of cancer cells have been established for testing the effi-
cacy of drugs. For example, lymphoma cancer cells were co-
cultured with neonatal stromal cells in a three-dimensional poly-
styrene scaffold to establish a blood cancer model for screening
various commercially available drugs [128]. Also, a “lung-on-a-
chip” microfluidic system was created with lung alveolar epithelial
cells and endothelial cells to study the drug treatment of respira-
tory diseases [129]. Drug toxicity was also studied using a co-
culture system [130], where hepatocytes were co-cultured with
other cell types such as ECs in the nylon scaffolds of a transwell
setting, thus creating a “three-dimensional liver” for testing the
effect of different drugs in inducing hepatotoxicity [130].
Furthermore, tissue modeling with co-cultures has been used to
study various diseases. For example, a co-culture of alveolar
epithelial cells, monocyte-derived macrophages, and dendritic cells
was established in a transwell system to model the alveolar
epithelial barrier and study infectious disease [131]. In addition, a
tissue model was built with breast cancer cells circulating through
microfluidic channels coated with ECs to study adhesion of these
cancer cells, angiogenesis, and how different chemokines can
stimulate cancer metastasis [132].
5. The role of biomaterials in the future of co-culture
research
Co-culture systems are often employed without much consid-
eration with regards to the potential influence of the substrate and
4472 K.G. Battiston et al. / Biomaterials 35 (2014) 4465e4476
its effect on experimental outcomes. In spite of this, it is well known
that the biomaterial surface plays an important role influencing cell
phenotype, proliferation, and other factors [45,133]. As an example,
the success of osteoblast:osteoclast co-culture systems has been
shown to be dependent on the biomaterial substrate (silk fibroin vs.
chitosan vs. PLLA) [134]. In a study by Jones et al., both silk fibroin
and chitosan supported growth in osteoblast and osteoclast mono-
and co-cultures, whereas PLLA showed comparatively poorer
osteoclast differentiation in both mono- and co-cultures. Synthetic
biomaterials thus have the potential to significantly advance the
field of co-culture research. By designing biomaterials with specific
properties, the biomaterials can be used to impart one cell type
with a desirable phenotype which in turn can influence the func-
tional response of the second cell type. Biomaterials scientists have
the ability to control numerous factors that can be used to optimize
a biomaterial substrate for a particular co-culture system. These
include surface chemistry, surface morphology, spatial patterning,
porosity, the incorporation of cell-responsive peptides, surface
wettability, electrical, and mechanical properties, amongst others.
The different properties can be varied to suit the specific needs of
different co-culture systems, depending on the aim of the experi-
ments and the different cell types involved.
5.1. Spatial patterning
The ability to pattern multiple cell types simultaneously by
providing a biomaterial substrate conducive to supporting a
particular geometric configuration allows for the control of devel-
oping tissue structure, as well as the ability to investigate cellecell
interactions in a controlled manner. While typical co-culture sys-
tems involve two cell types, the use of superhydrophobic structures
(using photoinitiated grafting of 2,2,3,3,3-pentafluoropropyl
methacrylate) has recently been reported to have the ability to
support patterns of up to twenty different cell types on one surface
[135]. Such systems may provide the necessary control for unrav-
eling some of the more complex interactions involved in co-culture
systems. Patterning has also been applied to three-dimensional
biomaterials through the use of photopatterned oligo(poly-
ethylene glycol)-fumarate:poly(ethylene glycol)-diacrylate hydro-
gels [136] using photomasks containing polygonal features, which
can be tailored to the needs of specific experiments. Furthermore,
substrates can be patterned to direct cell behavior, such as the use
of patterned PLG substrates for directed neurite extension in co-
cultures of neurons and accessory cells [100], as well as the
spatial control of neuron-glial cell co-cultures [137].
Another approach to spatial patterning involves the use of
temperature-sensitive substrates. Patterning culture substrates
with different thermo-responsive polymers has been used to pre-
pare patterned cultures of ECs and fibroblasts, with such patterns
potentially providing insight into cellecell communication phe-
nomena in co-culture systems [138]. In a study by Tsuda et al.,
thermo-responsive micropatterned surfaces using polyacrylamide
(PAAm) and thermally-responsive PIPAAm allow for the prepara-
tion of patterned endothelial cell sheets that can be layered with
fibroblast sheets. Microwell systems have also proven useful for
ESC differentiation studies and could provide useful information if
applied to co-culture systems [139,140]. While the use of bioma-
terial strategies to confine cells to particular geometries on two-
dimensional substrates provides significant promise in terms of
disentangling the effects of cellular cross-talk occurring in co-
culture systems, three-dimensional systems will be necessary for
recapitulating important structural features for tissue engineering
purposes.
Spatial patterning can also be achieved by patterning proteins
that are cell-type specific [141]. Such patterns have been produced
with features on the order of 100 mm. A particularly powerful
strategy involves the design of 3D matrices that allows for the
addition or removal of biological cues (ex. growth factors, extra-
cellular matrix ligands) in a bioorthogonal manner (i.e. not inter-
fering with biochemical processes in a living system) while cells
have already been growing on the biomaterial substrate (termed
‘4D matrices’) [142]. This allows for the biomaterial scientist to
temporally modulate the biological characteristics of the scaffold in
a controlled manner in order to facilitate a change in cell phenotype
when necessary.
Though not as controlled as site-specific patterning techniques,
control over a biomaterial scaffold’s porosity can also help to induce
specific effects. Scaffold porosity has been shown to be important in
regulating cell migration [143], diffusion rate, cell differentiation
(pre-osteoblasts) [144], and cell distribution (fibroblasts) [144].
Using PLGA-CaP scaffolds, Sicchieri et al. suggested the importance
of heterogenous pore size distribution for optimizing cell growth
and bone healing and demonstrated the importance of pore size in
promoting bone and blood vessel formation [145]. Porosity is thus
an important design strategy to consider, particularly for systems
requiring the promotion of vascularization [146].
5.2. Biomaterial mechanical properties
The elastic modulus of biomaterials is particularly important for
controlling stem cell differentiation. MSCs, for example, favor dif-
ferentiation to the osteoblastic lineage when cultured on stiff
substrates with modulus comparable to that of bone tissue [147].
Further control of a biomaterial’s mechanical properties, such as
introducing mechanical gradients within a surface, has also been
shown to have an effect on differentiation capacity, with narrow
mechanical gradients (70e90 kPa) inducing osteogenic differenti-
ation, whereas mechanical gradients with a broader range (10e
90 kPa) showed protein-dependent effects on osteogenic differ-
entiation, with inhibition observed on collagen vs. fibronectin
coated substrates [148]. These altered states of differentiation were
shown to be due to changes in paracrine signaling mechanisms,
which could take advantage of the upregulation of specific secreted
cytokines from one cell type, which is dependent on changes in
substrate properties, to elicit a desired response from another cell
type in co-culture.
Mechanical cues have also recently been reported to produce
self-patterning co-culture systems [149]. In a study by Palama et al.,
PDMS surfaces coated with laminin were prepared with areas
differing in stiffness. By creating stiff and soft regions on the PDMS
substrate, it was found that KU812 cells in the co-culture system
preferred stiff microdomains, while 3T3 cells preferred soft regions.
Patterning surfaces such as these with areas of varying stiffness
could allow for the spatial control and segregation for the growth of
different cell types, and provide useful design criteria when trying
to mimic tissue structures characterized by spatial patterning of
cells.
5.3. Surface functionality
Changes in surface functionality can be achieved through two
broad strategies: (1) the presentation of functional groups, with the
goal of presenting an optimized mixture of functional character or
surface wettability, and (2) the integration of cell-responsive pep-
tides and biochemical cues into the biomaterial to provide cell-
specific effects, such as the use of REDV peptides to target endo-
thelial cells [150]. General trends have been reported in the bio-
materials literature suggesting that certain surface chemistries
[133,151e153] or a specific range in surface wettability, are more
supportive of cell growth and attachment [133,151,154]. However,
 K.G. Battiston et al. / Biomaterials 35 (2014) 4465e4476
the literature is far from conclusive on specific chemical function-
alities and their ratios that are necessary for inducing targeted
biological responses from multiple cell types simultaneously. The
defined effects of certain functional groups are likely also influ-
enced by the combination of other factors, such as material me-
chanical properties. There is not likely a generalization that can be
applied to a single strategy for all types of synthetic and natural
biomaterials. A more strategic approach involves the use of specific
biochemical functionalities being incorporated into a biomaterial’s
chemical architecture [155,156]. Such approaches have been sug-
gested to be critical to the development of biomaterials for the
targeted differentiation of stem cells [156], and are a strategy that
will be key in developing biomaterials for co-culture systems.
Specific biochemical ligands can be chosen that have known
cell-specific effects. Such a strategy has been employed by Aizawa
and Shoichet to elucidate the role of ECs in governing retinal
stem and progenitor cell (RPSC) fate [155]. In the latter study, 3D
agarose-sulfide hydrogels were modified to contain a uniform
distribution of RGD and a concentration gradient of immobilized
VEGF. These hydrogels supported EC tubular-like morphologies,
while also demonstrating that ECs inhibit RPSC proliferation and
differentiation [155]. Similar targeting strategies have also been
employed to guide ECs in 3D scaffolds [157]. By providing such
guidance, EC distribution within the 3D scaffold can potentially
be controlled, allowing the scaffold to provide spatial patterning
of the ECs, while a second cell type, such as fibroblasts, can be
used to promote their survival or particular functional character
[3,146].
5.4. 2D vs. 3D culture systems
When trying to recapitulate cellular cross-talk that occurs in vivo
with an in vitro culture system, there are numerous issues that need
to be taken into consideration. Many co-culture systems are con-
ducted under 2D conditions, while tissues are inherently 3D and
contain several different topographical cues that cells can sense and
respond to [158]. As a result, when primary cells are isolated and
plated onto 2D substrates there is generally a loss of mature
phenotypic character and certain functionality, such as with he-
patocytes [36] and SMCs [159], which makes it difficult to mimic
the response of the cells in vivo. This loss of function provides an
opportunity in the context of biomaterial and co-culture strategies
to maintain cell function and phenotypic character, as has been
achieved by some in with primary hepatocytes, a cell type that is
notoriously difficult to maintain in culture [28,107,130]. The use of
3D culture systems in combination with an optimization of the
aforementioned biomaterial properties (surface functionality, me-
chanical properties, spatial patterning) will be important in the
next generation of biomaterials, whether they are developed to
mimic natural tissue structure or promote the growth and devel-
opment of new tissues in the lab.
6. Summary
Some of the issues facing co-culture experiments can be
addressed by including appropriate controls, while others require
proper experimental design and innovative methodologies for
retrieving maximum information from the co-culture systems
(Table 1). In all cases, however, it must be understood that while a
co-culture system can be optimized by varying seeding density, cell
type, and different media, the optimized culture conditions will be
specific to the biomaterial system which the cultures will be con-
ducted on. Different substrates will elicit different functional re-
sponses from cells which may alter the balance that was achieved
when the system was initially optimized on a different material. For
4473
this reason it is important that biomaterials be considered as a
more integral element in the experimental design of co-culture
systems, rather than the focus solely centred around the
biochemical reactions of the cellular components.
Acknowledgments
This review was supported from funding by the Natural Sciences
and Engineering Research Council (NSERC) Discovery (360520),
NSERC Alexander Graham Bell Canada Graduate Scholarship (CGS
D3), Canadian Institutes of Health Research (CIHR) Grant (230762),
Ontario Graduate Scholarship Program, and CIHR-Cell Signals
Training Fellowship program (TGF-53877).
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