Materials

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Advanced cell culture platforms: a growing quest

for emulating natural tissues
Cite this: Mater. Horiz., 2019,
6, 45
Marziye Mirbagheri,abc Vahid Adibnia,c Bethany R. Hughes,ab
Stephen D. Waldman,ab Xavier Banquyc and Dae Kun Hwang *ab
Published on 01 November 2018. Downloaded on 1/20/2019 9:00:43 PM.

Received 9th July 2018,
Accepted 1st November 2018
DOI: 10.1039/c8mh00803e
rsc.li/materials-horizons
In the body, cells inhabit within a complex three-dimensional (3D) extracellular matrix that provides
physical and chemical signals to regulate the cell fate. Cultured cells in Petri dishes and tissue culture
flasks (2D) receive completely different environmental cues compared to natural tissues, causing radical
alterations in cell morphology and function. Three-dimensional culture models have been able to
revolutionize biomedical applications by better emulating natural tissues. However, sample handling and
high-throughput screening can be challenging with 3D cell culture. Moreover, most 3D matrices are
unable to quantify intracellular mechanics due to their structurally undefined surface characteristics.
Therefore, highly structured surfaces (221 D) comprising various micro- and nano-patterns were
introduced to address these limitations. The topographical substrates have also been shown to retain
in vivo cell functionalities, such as proliferative capacity. Here, we review recent advancements in
modulation of surface patterns that have been able to control cell adhesion in two or three dimensions,
and their impacts on the cell behavior. Finally, we provide a comparison between 2D, 221 D and 3D
systems and present several clinical applications of non-planar substrates.

a
Department of Chemical Engineering, Faculty of Engineering & Architectural Science, Ryerson University, Toronto, Ontario M5B 2K3, Canada.
E-mail: dkhwang@ryerson.ca
b
Keenan Research Center, Li Ki Shing Knowledge Institute, St. Michael’s Hospital, Toronto, Ontario M5B 1W8, Canada
c
Faculty of Pharmacy, Université de Montréal, C.P. 6128, Succursale Centre Ville, Montreal, Quebec H3C 3J7, Canada

Marziye Mirbagheri obtained her Vahid Adibnia received his PhD in

BS and PhD degrees in Chemical
Engineering from the University
of Tehran and McGill University,
respectively. After completing her
PhD in 2017, as a postdoctoral
fellow, she joined the Advanced
Material Research Group at
Ryerson University and Li Ka
Shing Knowledge Institute at St.
Michael’s Hospital. She is now
continuing her postdoctoral
Marziye Mirbagheri training at the Faculty of
Pharmacy, University of Montreal.
Dr Mirbagheri has received several awards and fellowships during her
education and career. Her research interests mainly include
microfabrication and microfluidics, tissue engineering, organ-on-a-
chip, and nanoparticle interactions with hydrogels and biological
entities.
chemical engineering in summer
2017 from the Soft Matter &
Colloids Laboratory at McGill
University. As a postdoctoral
researcher, he then joined the
Biomaterials and Structured
Interfaces Laboratory at the
Faculty of Pharmacy of University
of Montreal. His research is focused
on nanoparticle interactions with
polymers, interfaces and complex
Vahid Adibnia viscoelastic media for drug delivery
purposes. He is also interested in
biomaterial engineering and tissue-mimetic polymeric structures. Dr
Adibnia has received several scientific awards and fellowships
including the McGill Engineering Doctoral Award (2012), McGill
Graduate Excellence Fellowship (2015) and FRQNT postdoctoral
fellowship (2018).

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1 Introduction
Cell culture experiments have been mainly performed on two-
dimensional (2D) platforms, such as multi-well plates, Petri
dishes, and tissue culture flasks, in which the cells are plated
onto a rigid planar surface. These conventional 2D substrates—with
ease of use and high cell viability—have notably improved our
understanding of basic cell function. However, they are notoriously
unable to appropriately imitate the cellular microenvironment
in vivo, leading to physiologically irrelevant cell behaviors in most
cases. Cells inherently respond to chemical (e.g., surface chemistry
and material composition) and physical (e.g., mechanical and
structural properties) signals from their surroundings at all length
scales,1–5 analyze the information and decide to undergo any of
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the cellular fate processes (division, apoptosis, migration and/or
differentiation).
Dr Waldman is a Professor of
Chemical Engineering at Ryerson
University, an Affiliated Scientist
at the Li Ka Shing Knowledge
Institute of St. Michael’s
Hospital, and the Director of the
Biomedical Engineering Graduate
Program (Ryerson University). He
was previously a Canada Research
Chair (2003–2013) and recently
named a Fellow of International
Orthopaedic Research (2016). His
Stephen D. Waldman research interests are primarily
centered on the development of
tissue engineered cartilages (articular cartilage, auricular cartilage,
and the intervertebral disc) with specific focus on the regulation of
mechanotransduction pathways, the effect of nutrient metabolism,
and biomaterial-cellular interactions to guide tissue growth.
Xavier Banquy is an associate
professor at the Faculty of
Pharmacy, Université de Montréal.
He has been the Canada Research
Chair in Bioinspired Materials and
Surfaces since 2013. His research
interests are divided into three main
streams: biomaterials development
and characterization of their inter-
actions with host tissue;
development of multifunctional
nanoformulations for cancer
Xavier Banquy treatment; fundamental research
in colloidal and surface science at
the biointerface.
46 | Mater. Horiz., 2019, 6, 45--71
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In their natural environment, nearly all cells reside within a
complex 3D fibrous meshwork, known as the extracellular
matrix (ECM).4,6 The 3D nanostructure of the ECM, which is
specific to each cell type, supports the cells and guides their
function.7 The loss of 3D cues, disorganized cell–cell inter-
actions, high oxygen tension, and high growth factor concentrations
in 2D cell culture can considerably alter the inter- and intra-cellular
behavior.8,9
The critical limitations of 2D cell culture can lead to several
practical complications. For example, in end-stage destructive
joint diseases, such as osteoporosis, osteoarthritis and bone
tumors, the bone quality and bone formation are decreased while
the bone resorption is increased. Current therapies involve the
replacement of damaged tissue by an implant. The life span of
these implants and, thus, the patients’ life quality greatly depend
on the initial bone tissue response.7 Surface topography is a key
parameter in controlling this initial response10 by modulating the
interactions between tissue and the implant.11 Many researchers
have already proved the beneficial effects of biomaterials that
mimic the bone surface roughness on osteoblast proliferation
and adhesion.12–18 The shortcomings of 2D planar platforms
have been repeatedly demonstrated in other studies as well.19,20
According to Lee et al.,20 the cytotoxicity testing of CdTe nano-
particles on HepG2 cells is dramatically different between 2D and
3D cultures, which can be problematic in preclinical drug
screening, where in vitro analyses determine the toxicity of a
target drug.21 Therefore, 2D planar platforms not only fail to
reproduce the 3D cellular microenvironment in the body, but
also can mislead our perception of cell responses.
To overcome these limitations, 3D cell culture methods
including spheroids, microcarriers, and tissue-engineered models
were introduced.22–33 While microcarriers are mainly used for
cellular expansion in vitro, spheroids and 3D scaffold-based
models have diverse applications in tissue engineering, cancer
research and fundamental studies of cellular function.34
Dr Dae Kun Hwang, Associate
Professor of Chemical Engineering
at Ryerson University since 2011, is
an applied materials scientist. He
is also a Canada Research Chair.
After obtaining his PhD degree in
Chemical Engineering from McGill
University in 2006, Dr Hwang did
his postdoctoral training at the
Massachusetts Institute of
Technology (MIT). Dr Hwang has
over ten years of research and
Dae Kun Hwang development experience in
advanced materials research using
microfluidics. His research focuses on the development of micro-
fabrication methods based on microfluidics, photo-lithography to
generate advanced functional materials of microparticles, membranes,
and wrinkled surfaces for biomedical applications.
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Materials Horizons
Spheroids are highly dense tissue analogs that are self-
assembled due to the tendency of adherent cells to aggregate.
Three-dimensional matrices are highly porous substrates that
can support cell function by homogeneously surrounding the
cell with extracellular materials.35 In tissue engineering, primary
cells from patients adhere and proliferate on the surface of a 3D
scaffold, generating the ECM components of the living tissue.
Cellular behavior in 3D cultures is relatively closer to natural
tissue, specifically, the cell morphology. For example, human
fibrochondrocytes have distinct 3D round or oval shapes in vivo
and on 3D scaffolds, whereas they exhibit stretched, fibroblast-
like morphologies in 2D culture flasks.36 Cell metabolism and
functionality, including proliferation, differentiation and gene
expression, are also significantly different between 2D and 3D
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models.19,34,36 This can be substantially important in clinical
applications, such as mesenchymal stem cell (MSC) transplantation
for treating osteogenesis imperfecta,37 where the loss of proliferative
capacity of MSCs in 2D culture can lead to poor engraftment and
limited cell survival upon transplantation into the body.38 Therefore,
3D systems are mainly favorable to 2D structures for cell culture and
tissue models. Nonetheless, several challenges remain.
With 3D cell culture, sample handling and imaging39 as well
as high-throughput screening35 can be challenging, demanding
technological innovations to fully benefit from the third
dimension.40,41 Furthermore, due to structurally undefined surface
characteristics, most 3D scaffolds are unable to quantify intra-
cellular mechanics.9 In recent years, highly structured surfaces
composed of various patterns, also known as 212 dimensional
(212D) objects, have been developed to promote our understanding
of cell behavior. On these substrates, adhered cells conform to
their specific surface patterns, whether simple, such as grooves, or
complex, such as plant topographies.42 This can significantly
improve drug testing and disease modeling, in which tissue
models should adequately represent natural cell organizations to
obtain physiologically relevant information.
This review highlights recent developments in modulation
of surface topography, and its impacts on cell behavior and
function. While most surface topographies induce 2D planar
cell morphologies, we also explain advanced 212D substrates
that have been able to control cell adhesion and cell shape in
three dimensions. Recall that here any topographical pattern
(e.g., groove, post, pit, and other complex designs) on a flat substrate
is recognized as 212D. Moreover, we discuss the advantages and
disadvantages of 212D substrates in comparison with 2D and 3D
models. Finally, we introduce several contemporary applications of
various non-planar cell culture platforms.
2 Cell culture on structured surfaces
Numerous soft and hard materials43–46 are utilized in biomedical
and healthcare industries for applications such as tissue
engineering,47 medical implants,48 and drug delivery.49,50
Particularly, many types of natural (e.g., collagen, alginate and
fibrin) and synthetic polymers (e.g., acrylamide, poly(ethylene
glycol) (PEG), poly(dimethyl siloxane) (PDMS), and poly(methyl
methacrylate) (PMMA)) have been used for tissue engineering
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and cell transplantation.51 For example, in a recent breakthrough,
European researchers were able to regenerate the entire human
epidermis using transgenic stem cells,52 providing high hopes for
treatment of the so-far incurable Junctional Epidermolysis Bullosa
(JEB) disease. In this research, the keratinocyte cells were cultured
onto fibrin gels, allowing for preparation of larger grafts from the
same number of clonogenic cells as would be needed for plastic-
cultured grafts.52,53
Such clinical studies are abundant and what they all have in
common is the importance of cell interactions with the scaf-
folding material, which can directly determine the cell response.
In recent years, it has been extensively documented that
surface topography in particular can greatly influence the cell
behavior.42,54–56 In this section, first we review topographical
substrates that lead to planar cell morphologies in which cells
migrate in a 2D plane. Next, advanced 212D structures that
induce a crossover from 2D to 3D cell behavior are discussed.
Here, in addition to physical patterning, surfaces may have been
treated chemically to control cell adhesion.44,57,58 Since the
focus of this review is on physical cues, the impact of chemical
surface modifications is discussed only when found relevant.
Moreover, throughout this article, the material types that are
used to construct the topographical substrates or 3D tissue-
engineered models are specified, as they can be important
factors in modulating cell–material interactions.59,60
Physical topographies that induce planar cell morphology
Diverse techniques including photo-,61,62,240 soft,63,64 colloidal65
and electron beam lithography,66 laser patterning,67 and dry
etching66,68 have been used to fabricate topographical surfaces
using various materials, such as polymers, silicon oxide, and
metals.69–71 Surface topography is especially important for
synthetic polymers due to the lack of biological recognition on
their surfaces.72
Schulte et al.73 demonstrated how topographical patterns
on PEG can solely manipulate cellular behavior without bio-
functionalization. According to their study, cells on smooth
polymer surfaces showed no pronounced stress fibre network
(Fig. 1(a)) with completely round bodies (Fig. 1(b)) and an
inclination to form small clusters (Fig. 1(c)).
However, on patterned surfaces with posts, protrusions were
formed (Fig. 1(d)), and the cell body flattened around the
surface structure (Fig. 1(e)) with a noticeably reduced cluster
formation. Similarly, single cells on top of (Fig. 1(f)) or within
(Fig. 1(g)) the grooves showed enhanced spreading and a larger
cell–surface contact area compared to smooth surfaces (Fig. 1(h)).
These observations indicated that fibroblast cells adhered and
spread on imprinted topographies, while the smooth surfaces
were nonadhesive.
The cell morphological and functional responses to surface
patterns greatly depend on the cell type74 as well as the pattern
type and dimensions (several examples are summarized in
Table 1). The effects of micro- and nano-grooves on different cells
have been widely investigated.75–78 On these linear cues, cells
usually align and migrate in the groove direction. However, the
cell response is strongly governed by the groove width and depth.
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Fig. 1 Mouse fibroblast cell line (L929) cultured on starPEG hydrogels with smooth surface after 24 h (a–c), post patterns after 4 h (d and e), and line
patterns with groove widths of 5 mm after 48 h (f and g) and 10 mm after 24 h (h) of incubation. Immuno-staining of F-actin (a, d and h) and cell nucleus
(a and h), and electron micrographs of dried cells (b, c and e–g). Scale bars: 5 mm. Reproduced with permission.73
Experiments with fibroblast show that groove width smaller
than the cell size provokes a stronger cell response,73 while
there is a cut-off value below which the cells no longer recognize
the patterns.77 For example, rat dermal fibroblasts obtained from
the ventral skin of male Wistar rats and cultured on nanogrooves
experience a threshold feature size of 35 nm.77 In contrast, cell
alignment has been shown to be proportional to groove depth
(Fig. 2), with an upper threshold above which no significant
improvements have been observed.62,73 For example, for MDCK
cell lines cultured on groove structures with groove width
B12 mm, the upper threshold for groove depth is reported to
be B1.1 mm.62 The groove depth has also been reported to be
more influential than its width.62,77
On a certain 212D substrate, the cell response may also vary
with time,77 emphasizing the need for sufficiently prolonged
culture time to establish the long-term cell behavior in medical
applications, such as implants. Guided cell alignment can be
crucial for disease modeling and drug testing, where cell
disruption due to a certain illness is analyzed to identify
potential drugs. Here, the model tissue should appropriately
represent the natural cell organization and function. In many
natural tissues, cells are elongated and structurally aligned.79,80
More clinical applications of these platforms are discussed in
Section 4.
Another topographical feature that has been used to inves-
tigate cellular behavior is micropillar or micropost. Similar to
grooves, the pillar size and spacing influence cell organization.
Ghibaudo et al.81 studied fibroblast migration onto chemically
identical substrates composed of micropillars. In this study, the
surfaces were uniformly coated with fibronectin to promote cell
adhesion, and the pillars were too stiff to be significantly
deformed by cell traction forces.82,83 Cell morphologies on
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pillars with a height of B2 mm were close to those on a flat
surface (Fig. 3(a)), while on taller pillars they adopted more
elongated and branched shapes (Fig. 3(b)). Whereas cells
migrated on top of the posts for small pillar spacings, they
encountered alternating flat and rough surfaces for wider
spacings (Fig. 3(c)). Furthermore, cell migration patterns were
changed from a fast and random movement on flat surfaces to
a slower and persistent movement on post arrays, allowing
their translocation from pillar to pillar. Compared to flat
surfaces, fewer actin stress fibers and focal adhesions were
observed, and they appeared more aggregated and more stable
over time on the pillars. This guidance of focal adhesion
formation was also observed in other studies.84,85 More information
on focal adhesions and their role in transmitting extracellular
physical or topographic signals can be found in ref. 85. A
potential application of these platforms is for cellular phenotype
discrimination, since various cell types react differently to pillar
topographies.
Studies of cellular mechanotransduction has also greatly
benefited from post patterns. Analyzing the cell responses to
physical cues suggests that when cells adhere to a surface, they
sense their environment by mechanically probing it.88 Adher-
ent cells exert contractile forces, also known as traction forces,
on the substrate using their actin–myosin cytoskeleton to
propel themselves.89–91 Traction forces regulate cell’s motility
and morphology,91 thus playing a crucial role in cell signaling,
proliferation, differentiation, migration and other biological
processes, such as angiogenesis, embryogenesis, inflammation
and wound healing. Specifically, assessing the contractility of human
stem cell-derived cardiomyocytes83 can help devise better strategies
for heart repair,92 disease modeling,93 and drug discovery.94 One of
the most important functional characteristics of a cardiomyocyte is
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Table 1 Examples of topographical patterns on planar surfaces and their impacts on various cell types

Surface Culture
Pattern Dimension (mm) Method Materials treatment Cell type time Effect Ref.
Grooves 5 o width o 10 Replica molding PEG — Mouse fibroblast 24 h Enhanced adhesion, spreading, and cell 73
Depth: 5 alignment and reduced cluster
Materials Horizons

formation.

Grooves 20 o width Replica molding PEG — Mouse fibroblast 24 h Cells reacted similar to those on a 73
Depth: 5 smooth surface.

Grooves 0.1 o width o1 Replica molding Polystyrene — Rat dermal fibroblast 4 (24) h Enhanced cell alignment. 77
0.075 (0.035)
o depth o0.35

Grooves Width: 6 Photolithography and PMMA — Baby Hamster Kidney — Poor cell alignment. 62
Depth: 0.56 dry etching fibroblast (aka BHK)

This journal is © The Royal Society of Chemistry 2019

Grooves Width: 6 Photolithography and PMMA — MDCK (epithelial cell) — B100% cell alignment. 62
Depth: 0.56 dry etching

Grooves Width: 6 Photolithography and PMMA — Baby Hamster Kidney — B100% cell alignment. 62
Depth: 2 dry etching fibroblast (aka BHK)

Grooves Width: 8 Hot-embossing imprint Polyimide Fibronectin Osteoblast — Cells aligned according to the 76
Depth: 4 lithography mechanical topography rather than
the chemical patterns.

Micropillars Height: 10 Photolithography and PDMS Fibronectin 3T3 fibroblast 6–24 h Cells on top of the posts had more 81
(undeformable) Diameter: 5 replica molding elongated and branched shapes, fewer
Spacing: 5 focal adhesions and slower movements.

Micropillars Height: 0.8 Photolithography and PDMS Fibronectin Malignant fibroblast 1–9 h As oppose to healthy cells, fewer cancer 111
(undeformable) Diameter: 1 replica molding cells were elongated and they exhibited a
Spacing: 1.6 disorganized motility.

Square micropillars Height: 4.5–14 Replica molding PLLA, TCPS, — C2C12 muscle cell 1–4 days Improved initial cell attachment but 112
(undeformable) Length: 5 PEOT-PBT, and reduced proliferation for hydrophobic
Spacing: 2–26 PDMSb materials (PDMS). However, better pro-
liferation on hydrophilic materials.

Nanopillars Diameter: 0.2 Nanoimprint UV resin — Human osteoblast 3 days Long filopodia extensions and 113
(undeformable) Spacing: 0.7 lithography lamellipodia formation.

Sharp-tip nanoposts Height: 0.05–0.6 Interface lithography Silicon — Human foreskin 3 days Elongated cells on medium height 97
Spacing: 0.23 and DRIE fibroblast features, but lower cell viability and
proliferation than on smooth surfaces,
which became more pronounced with
height. The cells also became smaller
and rounder with height.

Sharp-tip Height: 0.05–0.6 Interface lithography Silicon — Human foreskin 3 days Lower cell viability and proliferation 97
nanogrates Width: 0.23 and DRIE fibroblast than on smooth surfaces. Elongated cells
in the grate direction, with enhanced cell
alignment for taller grates.
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Table 1 (continued)

Surface Culture
Pattern Dimension (mm) Method Materials treatment Cell type time Effect Ref.

50 | Mater. Horiz., 2019, 6, 45--71

Nanogrates Height: 0–0.35 Nanoimprint photo- Poly(styrene), — Murine preosteoblast 24 h Physical topography induced higher cell 114
Pitch: 0.42 and 0.8 imprint and PMMA and a alignment than surface chemistry. Cell
lithography cross-linked alignment increased with grate height
dimethacrylate and trough width.

Hexagonal and Diameter: 0.12 Electron beam PMMA — Human osteopro- 21 days Enhanced osteogenesis compared to 98
disordered nanopit Depth: 0.1 lithography genitor and MSC planar surface, specifically on DSQ50
arrays Spacing: 0.3 arrays.a

Square array of Diameter: 0.12 Electron beam litho- PCL — Human MSC 28 days Prolonged maintenance of MSCs 99
nanopits Depth: 0.1 graphy and hot phenotype and multipotency, due to a
Spacing: 0.3 embossing reduced osteogenic differentiation but
improved MSC markers retention.

Orthogonal and Diameter: 0.035, Nanoimprinting and PMMA and PCL — Human fibroblast and — Reduced cell adhesion on ordered arrays 100
hexagonal array 0.075 and 0.12 hot embossing rat epitenon compared to planar surfaces.
of nanopits Spacing: 0.1, 0.2 and 0.3

Micropits Diameter: 7, 15 and 25 Photolithography Quartz — Human fibroblast 24 h Slightly improved cell proliferation, 103
Spacing: 20 and 40 and higher cell motility rates on 7 mm
Depth: 4.8 diameter pits. Cells can enter, divide in
and exit 25 mm diameter pits.

Square micropits Length: 2 Photolithography Quartz and Collagen on Neutrophil — Cell adhesion was stronger on quartz 104
Depth: 0.21 polyimide quartz than polyimide or collagen-coated
Spacing: 6–14 quartz. Cell motility was higher on
collagen-coated-quartz smooth surfaces.
Micropits with 10 mm spacing also
improved the motility.

Membranes with Diameter: 0.1–3 — Polycarbonate — Corneal epithelial cell 21 days Superior stratification on pores with 115
micro- and diameters in the range of 0.1–0.8 mm.
nano-sized
columnar pores
a b
Square arrays with dots displaced randomly by up to 50 nm on x and y axes from their position in a true square. Abbreviations are poly(L-lactic acid) (PLLA), tissue culture polystyrene (TCPS),
and a co-polymer of poly(ethylene oxide) and poly(butylene terephtalate) (PEOT/PBT).
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its ability to produce contractile forces, as mechanical contraction

Fig. 2 Scanning electron micrographs (SEM) of BHK cells on PMMA
microgrooves with 6 mm width and 300 nm (a) or 2 mm (b) depth. Cell
alignment improves with deeper grooves. Scale bars: 120 mm. Reproduced
with permission.62
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plays a key role in blood circulation throughout the body. For
example, measuring cardiomyocyte contractility can be of interest
in studying cardiac diseases associated with cardiomyopathies
(impaired contractility).
Microfabricated arrays of pillars have been extensively used
to manipulate and measure the mechanical interactions between
cells and their environment.82,83,95,96 Using silicon nanowires, Li
et al.86 compared the mechanical behavior of normal, benign and
malignant mammalian cells (Fig. 3(d)–(f)). According to their
results, cancer cells exhibit a larger traction force than normal
cells by 20 and 50% for the malignant and benign cells,

Fig. 3 (top) SEM images of fibroblast cell response to arrays of undeformable micropillars with height H, diameter D and spacing S. To promote
adhesion, the entire surfaces were coated with fibronectin: (a) H = 2 mm, D = 5 mm, and S = 5 mm; (b) H = 10 mm, D = 5 mm, and S = 5 mm; (c) H = 10 mm,
D = 10 mm, and S = 10 mm.81 (d) Schematic representation of cell adhesion to silicon nanowire arrays and the traction forces generated by cell’s actin
cytoskeleton. SEM images of normal mechanocytes (e) and cancerous L929 (f) cells bending silicon nanowires through their contraction forces. Scale
bars: 2 mm.86 (g) Graphical depiction of a finite-element method analysis of micropost with heights (L) deflected in response to a lateral traction force (F)
of 20 nN. (h) SEM micrographs of human MSCs plated onto PDMS micropost arrays with L = 6.10 (left) and 12.9 mm (right). For this study, only the post tips
were coated with fibronectin. Scale bars: 100 mm.87 Reproduced with permission.

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respectively. These comparative techniques can be beneficial
for disease diagnosis.
Intuitively, taller posts are more susceptible to bending in
response to a horizontal traction force. Experimentally and
numerically, Fu et al.87 demonstrated the relationship between
post height and deflection due to traction forces exerted by
human MSCs, and its consequent impact on cell behavior
(Fig. 3(g) and (h)). In contrast to soft (tall) microposts, cells
were well spread with highly organized actin stress fibers and
large focal adhesions on rigid (short) posts. Therefore, they
established a coupling between cell shape, focal adhesion and
cytoskeletal tension in response to substrate rigidity.
Pillar arrays in these studies had relatively flat tips. Choi
et al.97 investigated the behavior of human foreskin fibroblasts
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on sharp-tip nanoposts and nanogrates with various heights.
They observed a significantly lower cell viability and proliferation
on sharp-tip nanostructures compared to the smooth planar
surfaces, and this effect was more pronounced on nanoposts
versus nanogrates and with increasing height of both patterns.
Whereas cells elongated on medium height nanoposts, they
became smaller and rounder on taller posts, indicating poor cell
adhesion (Fig. 4(a)–(d)). Grate structures also provoked elongated
cell morphology in the grate direction; however, in contrast to
posts, cell alignment and elongation were more pronounced on
taller features (Fig. 4(e)–(h)). This is in agreement with previous
observations of enhanced cell alignment with groove depth (ridge
height).62,73
The last type of ordered surface topography is nano- or
micro-pit. Dalby et al.98 studied the interaction of two cell
types, osteoprogenitors and MSCs, when cultured on nanopits
of different symmetry, including orthogonal and hexagonal
arrays. Compared to cell culture on planar surfaces, human
osteoprogenitor cell density decreased, especially on hexagonal
arrays. Measuring the expression of bone-specific ECM proteins
by osteoprogenitors, namely osteopontin (OPN) and osteocalcin
(OCN), a slight increase in production of these proteins was
observed on symmetric pit arrays, with a larger difference for
Fig. 4 SEM micrographs of fibroblast cells cultured on smooth surfaces (a: control for posts, and e: control for grates), needle-like nanoposts (height
increasing from b to d), and blade-like nanogrates (height increasing from f to h). Insets show higher magnification of the sample’s nanotopography, and
arrows (2) in panels (f–h) indicate the nanograte directions. Scale bars: 50 mm. Reproduced with permission.97
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the square configuration (Fig. 5(a)–(c) and (f)–(h)). On the other
hand, increasing the randomness of nanopit arrangement
resulted in denser cell populations with increased levels of
OPN and OCN, specifically on disordered square arrays with
dots displaced randomly by up to 50 nm on x and y axes from
their position in a true square (DSQ50), as shown in Fig. 5(d),
(e), (i) and (j).
Similarly, MSCs cultured on DSQ50 exhibited intense cell
aggregation and early bone nodule formation with both OPN-
and OCN-positive regions. In contrast to osteoprogenitors, OPN
and OCN expressions by MSCs were lost on completely random
arrays (similar to the far-right, top panel in Fig. 5).
Later, McMurray et al.99 demonstrated how culturing cells
on orthogonal arrays of nanopits can allow a long-term maintenance
of MSC phenotype and multipotency, in contrast to cells cultured on
displaced arrangements such as DSQ50. In this study, cells on
symmetric orthogonal arrays retained expression of MSC markers,
i.e., STRO-1 and ALCAM (activated leukocyte cell adhesion molecule,
CD166), while no osteogenic markers (OPN and OCN) were
observed. Therefore, following a prolonged culture (28 days), MSCs
could be removed from the square pit arrays using trypsin, plated
onto a coverslip and treated with differentiation media to promote
osteogenesis or adipogenesis, indicating that cells not only expressed
MSC markers but also were multipotent. This again highlights the
impact of surface topography on differentiative capacity of MSCs
with crucial importance in cell transplantation therapies, where
tissue regeneration upon reinfusion depends on MSC differentiation
into tissue-specific cell types.
In another study, Curtis et al.100 reported that ordered
nanopit arrays (orthogonal or hexagonal) in polycaprolactone
(PCL) or polymethylmethacrylate (PMMA) can considerably
decrease cell adhesion compared to planar surfaces. As illustrated
in Fig. 6, cell adhesion on PCL surfaces with pit patterns was
very minimal, except for the smallest pits with diameter 35 nm.
Perhaps on these small patterns, cells ability to distinguish the
holes diminishes. A similar conclusion was drawn in previous
studies.101,102 Moreover, comparing the cells’ orientation on
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Fig. 5 OPN and OCN staining of osteoprogenitors after 21 days of culture. (top) Images of nanopits with diameter 120 nm, depth 100 nm, and absolute
or average center to center spacing 300 nm in hexagonal, square, displaced (DSQ50) and random configurations (respectively, from left to right).
Osteoprogenitors cultured on the control planar surface with lack of positive OPN and OCN (a and f); hexagonal array with poor cell adhesion (b and g);
square configuration with reduced cell density but slightly increased positive OPN and OCN compared to the control (c and h); DSQ50 showing bone
nodule formation and enhanced OPN and OCN expression (d and i); random arrangement with good cell population and improved OPN and OCN
production (e and j). Actin = red and OPN/OCN = green. Scale bar, as shown in panel b, is the same for panels a–j. Reproduced with permission.98

square or hexagonal pit arrays, they observed that not only were
the orientations nonrandom but they were also influenced by
different configurations, thus suggesting that cells are able to
distinguish between different symmetries.
Fig. 6 Rat epitenon cells cultured (24 h) on PCL substrates composed of
flat surfaces and pit patterns. In top, left corner of each image, diameter:center-
to-center spacing of nanopits is indicated. Cells are shown in black. Cell
adhesion on pit arrays is considerably lower than that on flat surfaces, with
relatively larger adhesion for smaller pits. Reproduced with permission.100
It is widely established that cell response can significantly
vary with length scale. While the aforementioned studies
analyzed cell behavior on nanopits, Berry et al.103 investigated
fibroblast attachment and motility on micropits. They reported
a clear dependence of cell response on pit size and spacing. Cell
proliferation slightly increased only on the smallest pits with
diameter 7 mm (a maximum difference of 6%), with even higher
proliferation on closer pit spacing. The largest pit diameters
(25 mm) allowed the cells to enter the pits while sustaining their
viability. Their results indicated that not only were cells able
to enter these pits, but they could also freely exit them, in
contrast to earlier observations for macrophages that were
trapped inside such pits.2 There was even evidence of cells
actually dividing inside the 25 mm pits prior to the daughter
cell emerging. On the smallest pits, however, a majority
of cells moved over the pits regardless of the spacing.
Furthermore, cells on the smallest pits moved faster, perhaps
by using the pit edges as footholds to gain mechanical
adhesion.104
A possible application of pit patterns is to establish appro-
priate pore sizes for 3D scaffolds in tissue engineering that will
enhance cell penetration (ingrowth)105 whilst maintaining
cell viability and proliferation. Microwells, if classified as pit
patterns with large pit diameters such that cells can colonize
inside the pits, have also been explored for high-throughput
studies of cancer progression and cancer drug discovery106–109
as well as for tissue repair and regeneration applications.110
A number of irregular topographies have also been the
subject of several studies. Among the most popular ones is
the surface roughness, which is usually quantified by measuring
the protrusions and depressions on a surface.55,116–120 A myriad
of evidence show that surface roughness can play an important

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role in cell function, for example increasing cell adhesion and

growth.121 Informative reviews are available on this subject.9,44
In an interesting work by Unadkat et al.,122 mathematical
algorithms were used to design a library of nonbiased and
random surface features on poly(lactic acid) with more than
2000 different topographies. As demonstrated in Fig. 7, a
multitude of MSC cell morphologies, including elongated,
branched, and round, was obtained. Using high-throughput
screening, they illustrated MSC proliferation and osteogenic
differentiation on the surface patterns.
The 212D platforms reviewed in this section induce different Fig. 8 SEM images of human corneal epithelial cells cultured on (a) nano-
cell morphologies, and furnish valuable information about cell grooved (width: 400 nm, depth: 600 nm, and ridge width: 70 nm) and (b) flat
metabolism and function compared to the oversimplified 2D control silicon oxide substrates,123 and (c) fibroblasts on a micropit-patterned
quartz surface (diameter: 25 mm and spacing: 40 mm).103 Reproduced with
planar surfaces (see Table 1 for more examples). Nonetheless, permission.
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cells on these substrates generally lay flat and migrate in a 2D

plane similarly to those on a smooth surface. Fig. 8(a) and (b)
demonstrates an example of such behavior by comparing the
human corneal epithelial cell shapes on a flat control and a
nanogrooved surface. The only exception is for micropits that
allow a certain level of 3D migration when cells enter the pits103
(Fig. 8(c)).
Devices that can capture more of the 3D complexity in
natural tissues are more desirable for understanding the cell
behavior in vivo. In the following, we will discuss novel 212D
platforms that have been able to induce cell movement and
morphology in 3 dimensions.
Advanced topographical substrates to induce 3D cell behavior
Various physical and chemical treatments have been employed
to create 212D scaffolds with more resemblance to topographical
complexity in natural ECM,124 and to manipulate cell adhesion
and shape in 3 dimensions.68,125,126
Using direct laser writing (DLW), Klein et al.127 fabricated a
substrate consisting of pillars that were connected by beams at
two different heights by 10 mm offset (Fig. 9(a)).
Poly(ethylene glycol) diacrylate (PEG-DA) crosslinked with
pentaerythritol tetraacrylate (PETA) was selected for the main

Fig. 7 Fluorescent microscopic images of spread and elongated MSCs, with alignments according to various topographical features (a–d). Actin stained
with Alexa Fluor 488 phalloidin = green, and nucleus stained with TOTO-3 = red. SEM images of cell morphologies on different topographies (e–h), and
round cells on two distinct platforms exhibiting different cell membrane textures (i and j). Scale bars: 90 mm (a–h) and 10 mm (i and j). Reproduced with
permission.122

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Fig. 9 (a) SEM image of a 221 D polymer scaffold composed of a protein-
repellent PEG-DA framework, which has embedded photoresist Ormo-
comp cubes on the PEG-DA beams (one cube is highlighted in red).
Primary chicken fibroblasts cultivated on the scaffold are immunostained
for fibronectin (red) and f-actin (green). Top view (b) and 3D construction
(c and d) of confocal image stacks illustrating adherent cells to one or
more fibronectin-positive Ormocomp cubes at the same or different
heights. Reproduced with permission.127
skeleton of the scaffold, as PEG-DA is known for its protein-
repellent properties that prevent cell attachment. To control
cell adhesion and ECM distribution, protein-binding Ormo-
comp cubes were embedded on PEG-DA beams. Ormocomp is a
biocompatible photoresist. Upon incubation of these scaffolds
with fibronectin, this ECM protein bound preferentially to the
Ormocomp cubes, as shown by immunofluorescence labelling
in Fig. 9. Therefore, chicken fibroblasts cultured on these
platforms adhered and spread on a single or multiple Ormo-
comp cubes, particularly revealing a true 3D growth pattern
when spreading between cubes on beams at different heights
(Fig. 9(c) and (d)).
Greiner et al.128 studied the growth of several cell types on
fibronectin-coated 212D platforms fabricated also using DLW
(Fig. 10 (left)). As illustrated in Fig. 10, the cells adapted true 3D
geometries on these platforms. It was also demonstrated that
cell and nuclear volumes may change between 2D and 3D cell
morphology, depending on the cell type. Whereas epithelial
cells retained similar cell and nuclear volumes regardless of the
cell morphology, fibroblasts displayed significantly increased
cell and nuclear volumes for the 3D cell shapes compared to the
2D. They attributed these differences to the tissue-specific cell
arrangements in vivo, where fibroblasts reside within loose
connective tissues and epithelial cells within densely-packed
flat layers. Nevertheless, the nucleus-to-cell (N/C) volume ratios
remained constant for all cell types and culture conditions. N/C
ratio alteration is a widely used metric in cancer detection.129
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In the aforementioned studies, an ECM protein (i.e., fibronectin)
was used to control binding sites and, therefore, manipulate cell
adhesion to induce 3D morphologies. Using a similar strategy,
Ochesner et al.68 were able to provide a 3D shape control of single
cells in microwell arrays. In their study, cell adhesion was limited
within the microwells by passivation of the upper flat surface, while
backfilling the wells with adhesive proteins or lipid bilayers.
Another creative use of adhesive proteins for cell adhesion
was demonstrated by Richter et al.,130 who produced 212D cell
instructive platforms by incorporating two distinct ECM binding
proteins in a passivating framework. They illustrated that cells
with different sets of integrin receptors preferentially adhered to
one of the proteins. However, since the binding sites were in the
same height, cells spread in a 2D plane. Positioning the distinct
binding proteins at different levels can possibly be a strategic
approach to obtain 3D cell shapes.
In a recent work by our group,131 surface wrinkling was
implemented as a physical technique to precisely distribute
adhesion sites and induce 3D cell morphologies. Using photo-
lithography and microfluidics, arrays of PEG-DA posts were
created. By controlling the UV exposure time, a thin layer of
partially cured polymer was formed on the posts, which wrinkled
when exposed to plasma. The wrinkling mechanism is extensively
explained by Kim et al.132 Whereas fibroblast cells adhered to the
flat surface when the posts were smooth, they preferentially
conformed to the wrinkled posts exhibiting 3D morphologies
(Fig. 11(a) and (b)). It has also been shown in previous studies
that cells prefer the flat surface in arrays of smooth pillars.81
Interestingly, some cells on adjacent wrinkled posts appeared to
form bridges, as shown in Fig. 12(c).
In contrast to 212D platforms reviewed in the previous section,
studies on advanced surface topographies that are capable of
producing 3D cell geometries have just begun in the past decade.
Nonetheless, they have provided exciting opportunities to elucidate
the cell metabolism and function in more detail, and help
advance engineered microenvironments to better emulate in vivo
conditions.
3 A comparison with three-dimensional
cell culture systems
In this section, we first briefly introduce different 3D cell
culture techniques and explain their differences with 2D mono-
layer models. Next, the advantages and disadvantages of 3D cultures
are weighed against 212D platforms for human bone-marrow-derived
MSCs, as one of the most investigated cell types using these
techniques in the literature.
3.1 3D vs. 2D (planar surfaces)
Three-dimensional ex vivo cell culture models can be categorized
as spheroids or microtissues, microcarriers, and tissue-engineered
models. Microcarriers are mainly used to expand anchorage-
dependent cells in vitro, providing several advantages compared
to 2D models such as enhancing in vitro differentiation30 with high
reproducibility and scale-up potential.31 However, due to their
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Fig. 10 Buffalo rat liver cell growth on a 2D control surface (a), and a stiff (b) and a soft (c) 221 D scaffold, which were all coated with fibronectin to control
cell adhesion. Left panels indicate the SEM images of culturing structures, the middle panels present the maximum projections of confocal image stacks,
and the right panels are 3D constructions of confocal images. F-Actin = green and nucleus = blue. Reproduced with permission.128
Fig. 11 SEM images of bovine fibroblast cell growth on smooth (a) and
wrinkled (b and c) posts. Comparing (a and b), cells preferred the flat surface
when posts were smooth, whereas with the wrinkled posts they attached to and
spread on the posts, conforming their 3D curvature. Some cells even formed
bridges between the wrinkled posts, creating an overhanging connection
between them (c). Scale bars: 30 mm. Reproduced with permission.131
relatively less diverse applications, we will mainly focus on spheroids
and 3D tissue-engineered models in this review.
Cellular spheroids are simple 3D tissue analogs that are self-
assembled due to the tendency of adherent cells to aggregate.
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The term spheroid is more commonly used because most tissue
aggregates are spherical, whereas a more general term, namely
multi-cellular microtissues or simply microtissues, describes
other shapes as well.133,134 Several single culture or co-culture
techniques, such as pellet culture, spinner culture, hanging
drop, rotating wall vessel (RWV) and microfluidics, have been
used to create microtissues.32,33 Regardless of the fabrication
method, spheroids with a high cell density create more in vivo-
like microenvironments by forming complex cell–cell and cell–
ECM interactions in addition to establishing diffusion barriers
to soluble components (e.g., nutrients, oxygen, growth factors
and wastes). These biomimetic characteristics are particularly
crucial for cancer drug discovery applications. The molecular
gradient of nutrients and oxygen can cause necrosis and
hypoxia toward the center of the spheroids, and the enhanced
intercellular communications can regulate cell function and
fate.32 Similarly, solid tumors often show hypoxia/necrosis,
slow proliferation and drug diffusion barriers that contribute
to cancer drug resistance.135 These features are not properly
reflected in monolayer cell cultures in which cells proliferate
rapidly, show high drug activity and have equal access to high
concentrations of nutrients and oxygen.
Several other studies have demonstrated the benefits of
spheroids over 2D monolayers.136–138 For example, as illustrated
in Fig. 12, hepatocyte spheroids exhibit liver-like functionalities
with improved differentiation and reduced proliferation in
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Fig. 12 Histological examination of human liver and HepG2 spheroids. Scale bar: 100 mm. Reproduced with permission.139
contrast to 2D monolayers of hepatocytes, which rapidly
dedifferentiate and lose critical hepatocyte functions.139
Nonetheless, various challenges in spheroid culture procedures
still remain. Conventional methods for spheroid production, such
as spinner cultures, hanging drop, and rotating systems, can be
labor-intensive, time-consuming, and are unable to maintain
spheroid size uniformity on large scales,140,141 whereas more
sophisticated platforms142 can be more complicated and
require specially trained users for fabrication and operation.
Table 2 provides a list of advantages and disadvantages of
spheroids compared to 2D models.
In 3D tissue-engineered models, cells are grown in 3D matrices
or scaffolds that are prepared ex vivo using natural and/or synthetic
materials. Generally, it is preferable to use the materials extracted
from living tissue ECM that are specific to the cells under study.
Commercially available gelatinous protein mixtures, such as
Matrigels, and decellularized tissues used for treating cardio-
vascular diseases165 and diabetes166 as well as for reconstruction
medicine167 are practical examples of this method. However,
such matrices are often difficult to customize and may vary from
batch to batch.158 Alternatively, attention has been paid to
synthetic polymers that can form 3D networks with microscopic
pores. Polymer selection depends on cell adhesion, biocompatibility,
wettability and structural properties such as porosity, pore geometry,
transport and degradation kinetics. Detailed reviews on artificial 3D
cell culture scaffolds and their characteristics can be found
elsewhere.4,39,158,168–174 Spatial organization of cells in the media
directly depends on their microstructural geometry. Therefore,
perfecting the material structure to mimic the tissue is a key step
in regulating cell growth and promoting the functionality of specific
cell types to produce targeted outcomes that are unachievable in 2D
planar models.
An example of artificial 3D scaffolds that direct cell pro-
liferation and differentiation is a 3D self-assembled network of
peptide amphiphiles introduced by Silva et al.,175 which was
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used to grow murine neural progenitor cells. The self-assembly
was triggered by injection of cell culture medium, causing the
peptides to aggregate into nanofibers with high aspect ratios,
thereby forming a solid hydrogel that mechanically supported
the medium (Fig. 13(a)). The molecular design of the scaffolds
promoted neurite sprouting and direct neurite growth, inducing
very rapid cell differentiation into neurons.
Alginate-based scaffolds have been used to promote directed
axon regeneration in vitro and in adult rat spinal cord lesions
in vivo.155 The hydrogel microstructure contained self-assembled
anisotropic capillaries that were formed during the gel synthesis
(Fig. 13(b)). After seeding with adult neural progenitor cells,
which are known to improve axon regeneration, these gels were
implanted into acute cervical spinal cord lesions in rats and
integrated into the spinal cord parenchyma without inflammatory
responses. In accordance with in vitro findings, the anisotropic
structure of these implants induced directed axon regeneration
across the artificial scaffold, as shown in Fig. 13(b)(V) and (VI).
Moreover, the combined cell- and artificial scaffold-based therapy
further improved neuron restoration. The alginate-based hydrogels
were presented as a promising strategy for nerve regrowth
following spinal cord injury, when the targeted neuron rein-
nervation depends on longitudinally directed regrowth of
transected axons.
Tumor microenvironments are temporally and spatially
heterogeneous, and it is unclear how the gradations in biophysical
cues can impact malignant phenotype and response to therapy.
Three dimensional platforms capable of mimicking this complexity
can provide valuable information about tumor etiology, growth and
spreading. In an attempt to replicate the heterogeneity of tumor
ECMs, Pedron et al.156 generated 3D hydrogels with controlled
spatial gradient of matrix and cellular content (Fig. 13(c)).
Investigating glioblastoma multiforme (GBM), as a common
form of brain tumor in adults, they demonstrated how gradients
in cell density can lead to heterogeneous gene expression.
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Table 2 Key differences in cellular characteristics and processes between 2D and 3D cell culture models
Cellular
characteristics 2D monolayer
Morphology Flat and stretched
monolayer
Adhesion Cells adhere to the
supporting surface
Viability Cell viability is acceptable
Stage of cell cycle Cells are more likely to be
at the same cycle due to
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being evenly exposed to the
medium
Proliferation Often proliferate faster
than in vivo
Differentiation Often reduces the
differentiative capacities
of stem cells
Gene and protein Gene and protein
expression expression levels generally
differ from in vivo models
Cellular Cell–surface interactions
interactions prevail rather than cell–cell
and cell–ECM interactions
Directed growth Cells grow randomly
Sample handling Simple and well suited for
and imaging high-throughput screening
Scale up and Less expensive for large-
reproducibility scale studies and easier to
reproduce
Complexity Involve the simplest
procedures
Drug testing Drugs appear effective
but the results are
physiologically irrelevant
Wound healing Lower effectiveness
compared to 3D models,
as simulating natural
conditions is almost
impossible
Moreover, they reported that a spatially graded matrix stiffness
can significantly alter cell proliferation and morphology.
Several other novel hydrogel designs for cell culture have
been reported in the literature176–182 that demonstrate the
superiority of 3D cultures to 2D planar substrates. Table 2 also
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3D cell spheroids
Aggregates of natural 3D shapes at
the interior of spheroid
Cell-to-cell adhesion is dominant
Cell viability is improved or is
similar to 2D
May contain proliferating,
quiescent, hypoxic and necrotic
cells. Larger spheroids show more
necrosis in the center due to
hypoxia
Lower or comparable proliferation
rate compared to 2D
Can enhance the differentiation
potential of multipotent cells
Gene and protein expression
profiles are closer to natural
tissues
Cellular and cell–ECM
interactions are promoted
Cellular aggregates are mainly
spherical, whereas other shapes
were obtained in limited studies
Sample handling is more
complicated while large number
of spheroids can be provided for
high-throughput drug testing.
Depending on the fabrication
technique, spheroid properties
can be reproducible or greatly
variable
Often labor-intensive,
time-consuming, more
expensive and more complicated
Cells often show more resistance
to therapeutics and better
represent in vivo conditions
Spheroids have been shown to
promote angiogenesis and wound
healing compared to single cells
recovered from 2D cultures
furnishes a thorough comparison between 3D scaffold-based
and 2D monolayer models. Ultimately, an ideal environment
for 3D cell culture requires a control of structural and physico-
chemical properties on length scales from nano to macro, and
time scales from seconds to weeks. Therefore, the next step in
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3D tissue-engineered models Ref.
Can assume stellate, round or elongated 135, 139,
morphologies. Regardless of the shape they 143 and
are homogeneously surrounded by the ECM 144
Both cell–cell and cell–ECM adhesion can be 144 and
observed 145
3D cultures can significantly increase cell 32, 136,
viability 137, 139,
146 and
147
— 139
Lower, comparable or higher proliferation 139, 143,
rate compared to 2D 148–150
Can preserve the differentiative capacities 34, 136,
and provide a more reliable understanding 151–153
of differentiation mechanisms
Can furnish similar gene and protein 34, 139
expressions as in in vivo conditions and 154
Interactions of the cell cytoskeleton and the 34, 138
ECM can significantly vary between 2D and and 139
3D scaffold-based models, impacting
various cell activities, such as apoptosis
Can be customized to direct cellular growth 133, 134,
in various patterns, such as elongated or 155 and
heterogeneous cell distributions 156
Sample handling and high-throughput 35, 39 and
screening is more challenging 157
Reproducibility can be challenging and 135, 139,
scaffold’s properties may vary from batch to 141, 158
batch for naturally derived scaffolds and 159
More expensive and more complicated 141
compared to 2D
Cells responses are more reliable, showing 135 and
more resistance to drugs or providing better 160–162
prediction of drug cytotoxicity and efficacy
These models can allow a more reliable 163 and
studying of wound healing by better 164
evaluating the cell contractility and matrix
compaction as well as simulating the
mechanical forces in tissue in vivo
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Fig. 13 (a) (I) Schematic illustration of the self-assembly of peptide amphiphile molecules into nanofibers. (II) SEM micrograph of the nanofiber network
after adding the cell medium. (III) Macroscopic image of the gel formed by adding peptide solution to the cell culture medium.175 (b) (I) Illustration of
anisotropic capillary alginate gel formation. (II) Macroscopic image of the gel. (III) Cross section of the gel. (IV) Longitudinal section of the gel. (V and VI)
Confocal fluorescent micrographs show regenerating axons in the alginate capillaries. Scale bars: 1 cm (b, II), 100 mm (b, III and IV), and 50 mm (b, V and
VI).155 (c) (I) Schematic illustration of gelatin hydrogels designed by microfluidics that contain a gradient in cell number. (II) Increase in hypoxia inducible
factor 1 gene expression (green) with increasing cell (blue) density across the gel. Scale bar: 50 mm.156 Reproduced with permission.

effectively mimicking the ECM and guiding cell behavior is to
develop materials whose properties can be tuned according to
the time and length scale of the cell development. The conventional
fabrication approaches, such as lithography, molding and solvent
casting, are unable to sufficiently control the scaffold properties.
These include nanoscale surface modifications, such as biofunctio-
nalization; microstructural details, such as pore size and shape,
porosity, and pore distribution; macroscale architecture, such as the
external appearances of artificial devices. Hence, a user-controlled
and replicable technique seems essential. 3D bioprinting has
recently emerged to address these shortcomings.
Bioprinting is a rapidly growing computer-aided technology
that can create complex tissue architectures by patterning a cell-
encapsulating bioink.183 Owing to its significantly improved reso-
lution and cheaper cost in the recent years, it is implemented in
tissue engineering to create 3D cell-laden materials in which the
bioink contains the cells,184,185 or to produce scaffolds onto which
cells are seeded.186 Due to its versatile patterning of cells and
scaffolding materials, bioprinting has been able to revolutionize
biomedical applications, such as organ-on-a-chip devices,187
cancer research studies,188,189 and pharmaceutical analyses.190
Organ printing, which uses this technology to prototype living
human organs (Fig. 14), offers new promise for solving organ
transplantation crisis.191–194
Bioprinted structures offer various desirable features to the
field of tissue engineering. Spatial patterning of complex 3D
environments can be relatively easy using 3D printing.183,198
Bioprinting also allows for co-culturing of multiple cell-types,
which can be attractive for 3D modeling of neural tissues185,199
that contain various glial cell types.183 Moreover, it is known for
its ability to control the scaffold porosity and produce inter-
connected pores,200 which can greatly enhance cell attachment and
ingrowth,201 both desirable in tissue engineering applications.202,203
For more information about different aspects of bioprinting, such
as the choice of material and bioprinter, challenges, and future
perspectives of this technology, the reader is encouraged to see
detailed reviews available elsewhere.183,185,194,204–206
3.2 3D vs. 212D (non-planar surfaces)
Two-dimensional monolayer cultures are usually used as control
experiments when developing 3D culture systems and investigating
their impacts on cellular functions. Therefore, direct comparison

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Fig. 14 A 3D bioprinter (a) was used to prototype a human ear (b).195 (c) Image of a 3D printed bionic ear during in vitro culture. Scale bar: 1 cm.196 Panels
(d, e and f) show a heart valve model designed by Solidworks, the bioprinted valve conduit, and the cross-sectional view of live/dead cells from surface to
4300 mm depth, respectively.197 Reproduced with permission.
between 2D and 3D models, reported in Section 3.1, could be readily
found in the literature. However, topographical substrates are not
usually directly compared with 3D systems in a single study,
requiring a broader and more careful survey to acquire this
comparative information. In addition, an accurate comparison
can be conducted only between similar cell types that are
derived from the same tissue origin. For example, Baksh
et al.207 demonstrated that human MSCs derived from umbilical
cord and bone marrow may exhibit different proliferative and
differentiative capacities. Therefore, we will only focus on bone-
marrow-derived human MSCs in this section, since they have
been more extensively investigated using all culture systems.
Bone-marrow-derived MSCs are multipotential cells which
are capable of self-renewing and differentiating to a number of
cell types, including osteocytes, chondrocytes, adipocytes,
hepatocytes, myocytes, neurons, and cardiomyocytes. For tissue
engineering applications, MSCs are often expanded and/or
differentiated in vitro prior to in vivo transplantation. Conventional
2D monolayer methods normally fail to maintain and/or support
efficient cellular functions such as differentiation and replication.
On the other hand, topographical substrates, spheroids and 3D
tissue constructs have been shown to improve MSC differentiation
while providing proliferation and viability similar to or higher than
2D models, according to several studies summarized in Table 3.
However, the cell characteristics as well as biomaterial properties
can greatly impact the outcome. For example, 3D-polycaprolactone/
hydroxyapatite (PCL/HAp) scaffolds have been shown to increase
the proliferation of bone-marrow-derived MSCs compared to 2D
monolayers, whereas adipose-derived MSCs exhibited similar
replication rate on 3D scaffolds and monolayer cultures.208 In
another study, MSC viability on nanofibrous topographies with
random fibre orientations, which was assumed to better mimic
the structure of the native bone ECM, was higher than that on
aligned fibres.209
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Comparing 212D and 3D systems, spheroids might accom-
modate higher cell culture capacity in cell aggregates than 212D
substrates and 3D scaffolds with limited culture spaces.212
However, the biomaterial-based culture methods often improve
cell proliferation (Table 3), while spheroids usually yield similar/
lower proliferation rate compared to 2D monolayers.136,139,211
On the other hand, MSC differentiation can be improved using
all three methods, thus, the optimal selection should be based
on the final application. Due to their structurally controlled
surface characteristics, topographical substrates can provide
more detailed information about intercellular mechanics by
tailoring and manipulating cell–cell interactions. Similarly, they
may be more desirable for high-throughput screening and
imaging, since cells are far more accessible for optical char-
acterization when they are plated onto a surface than confined
within the pores of a 3D scaffold with high thickness.
While both spheroids and scaffold-based models can be
good sources for obtaining a supply of specifically differentiated
MSCs or providing valuable information about MSC differentiation,
one might be more desirable than the other depending on the
purpose of the culture. In tissue engineering applications, such as
cartilage repair, spheroids usually suffer from small size and
uniformly weak mechanical properties,213 and these limitations
can be addressed using 3D scaffold-based models. Spheroids,
however, have been rather more attractive for other applications
such as cancer research by better mimicking the human solid
tumor microenvironments, where necrotic, quiescent and proliferat-
ing cells are present.34 In some cases, a combination of both
methods can be more effective. For example, hydrogel encapsulated
spheroids have been shown to exhibit greater resistance to apoptosis
than entrapped dissociated cells.136 Nonetheless, a judicious
selection of the cell culture method is a trade-off between
the efficacy and performance of the model, sample handling,
complexity, reproducibility and economical factors.
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Table 3 Bone-marrow-derived MSC cellular functions in 221 D and 3D cell culture methods in comparison with 2D models

Structure/fabrication Cell morphology Viability Proliferation Differentiation Ref.

212D substrates
Square array of PMMA Flat and polygonal — Higher proliferation Higher osteoblastic 98 and
nanopits morphology and cell phenotype/ differentiation on displaced 99
multipotency retention arrays than 2D flat and
on ordered arrays than ordered nanopits
2D planar control and
displaced nanopits

Silicon nanowires More spherical and less Cell survivability — Shortest nanowires significantly 210
elongated morphologies was close to promoted osteogenic
with sturdy protrusions monolayers differentiation than 2D mono-
layers and longer nanowires

PCL nanofiber Aligned according to Higher viability on Improved proliferation on Osteochondrogenic 209
substrates fibre orientation. Cells random fibres than random fibres compared differentiation was significantly
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on aligned fibers were aligned fibres and with aligned orientations increased on random fibres
elongated and spindle- 2D culture plates and monolayers
shaped, whereas they
were mostly round on
random-oriented fibers

3D cell spheroids
Spinner flasks and Continuous layer of Comparable 4.3% or 3.5% proliferating Increased osteogenic and 211
RWV elongated MSCs on viability to cells for spinner and RWV, adipogenic differentiation
the outer surface with monolayer cultures which was comparable to in spheroids, where the latter
irregularly shaped cells monolayers was more significant in RWV
in the spheroid interior

Hydrogel encapsulated — Entrapped spheroids Proliferation did not differ Similar osteogenic potential 136
spheroids formed using exhibit greater between entrapped for spheroids and their
the hanging drop resistance to spheroids and dissociated monolayer counterparts, when
technique apoptosis than cells encapsulated in fibrin gels
entrapped cells from
monolayer cultures

Photolithography and 3D cell aggregates High viability was — Adipogenic and osteogenic 212
micropatterning obtained for at least differentiation was more
techniques 30 days efficient in spheroids than
monolayers

3D tissue-engineered models
3D PCL nanofibrousa Flat fibroblast-like cells on — Cell proliferation was Chondrogenic differentiation 213
scaffolds the surface with round, higher than in cell pellets was similar to cell pellets
chondrocyte-like cells in
middle, surrounded by
abundant cartilaginous
matrix

b-Tricalcium phosphate — Mean values of Proliferation was 3D culture provided higher 150
(TCP) ceramics metabolic activity proportional to porosity osteogenic differentiation
was larger with in 3D scaffolds, and than 2D monolayer, but it was
porosity, however, significantly higher independent of porosity
the comparison was than monolayer culture
elusive due to high
standard deviations

3D-Polycaprolactone/ — Cells were highly Higher proliferation rate ALP activity and mineral 208
hydroxyapatite (PCL/ viable than 2D monolayer deposition were higher on the
HAp) scaffold 3D scaffold than 2D culture
plates, indicating higher
osteogenic differentiation
a
Cell functions in this example are in comparison with their spheroid counterparts.

4 Applications 4.1 Cell therapy

Cell-based devices, analyses and therapies are increasingly being Cell therapy has been widely used in a variety of clinical
used in clinical applications. Here, we describe some recent contexts. Stem cell transplantation is a promising therapeutic
examples, although many others can be found in the literature. strategy for several illnesses, such as neurological214 and

This journal is © The Royal Society of Chemistry 2019 Mater. Horiz., 2019, 6, 45--71 | 61

Review
cardiovascular215 diseases, type 1 diabetes,216 and osteogenesis
imperfecta.37,217
Prior to implantation, the stem cells isolated from a donor
are expanded in vitro using conventional 2D tissue culture
techniques.218 However, it has been demonstrated that certain
cell-specific properties of stem cells are lost over time in 2D
culture to the point they no longer reflect in vivo cell behavior.38,219
These cells are usually unable to maintain long-term tissue
regeneration upon reinfusion into the body, highlighting the
necessity of improved methods for in vitro cell culture. For
example, 212D99 and 3D211 cultures have been shown to enhance
osteogenic and adipogenic differentiation potential in MSCs
when compared with 2D monolayer cultures.
Recently, 3D scaffolds were used to regenerate an entire,
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fully functional epidermis on a seven-year-old child suffering
from a life-threatening form of JEB.52 For this treatment, the
keratinocyte cells were cultured on 3D fibrin scaffolds, allowing
the preparation of larger grafts from the same number of cells
as would be needed for 2D plastic-cultured grafts.52,53
4.2 Vaccine production
In 1949, the key scientific discovery by Enders et al.220 that
poliovirus could be grown in cell culture led to the development
of poliovirus vaccine, and to significant advances in producing
other virus vaccines in the following years. Prior to this, the few
available vaccines were produced in animal systems, such as
embryonated eggs for influenza and yellow fever viruses.221
Currently, many viruses are still being produced in animal
systems due to reliably high yields. However, these methods are
time consuming and expensive. In addition, in some cases,
such as embryonated eggs for seasonal influenza, the vaccines
are developed more than 6 months in advance of the flu season
to allow for adequate time to produce the required quantities.
This lag can be problematic if the selected strains do not match
the predominant ones during the winter. Unfortunately, people
with egg allergies cannot use vaccines that are produced in eggs.
Therefore, finding an alternative approach, such as in vitro cell
culture, can be attractive.222 In this regard, Tree et al.223 introduced
the 3D culture of MDCK cells using microcarriers224 as a potential
technique for producing influenza virus A vaccine strains.
Although they suggested that a comparable yield of influenza virus
A production could be obtained between scaled-up 3D cell cultures
and embryonated eggs, the differences may be larger for a different
strain of influenza virus. Thus, the search for an efficient approach
is still ongoing.
4.3 Disease modeling
Engineered tissues can furnish a powerful tool to probe the
disruption in cell organization and function in response to a
disease.225 However, to obtain biologically relevant models, the cell
arrangement should be closely related to the tissue organization
in vivo. For example, human embryonic stem cells can be directed
into cardiac lineages, such as cardiomyocytes,226 for disease
modeling. Whereas ventricular cardiomyocytes are naturally
aligned, human embryonic stem cell-derived cardiomyocytes are
heterogenous and randomly organized. It has been demonstrated
62 | Mater. Horiz., 2019, 6, 45--71
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Materials Horizons
that biomimetic culture platforms comprising physical topo-
graphies, such as wrinkles227 and grooves,228 can stimulate
cardiomyocyte cell alignment and, therefore, lead to physio-
logically relevant responses.228
The dynamic cell alignment can be an important factor
when modeling the progression of various diseases. For example,
during heart failure, the highly aligned cardiac muscle fibres
become disorganized, causing progressive fibrosis and ventri-
cular dilatation. While many of the in vitro models using surface
topographies are static, a tunable culture platform that can
dynamically manipulate the temporal and spatial cell align-
ments can be attractive. Lam et al.229 were the first to introduce
reversible cell alignment by compression-induced dynamic
lamellar patterns on PDMS substrates. However, their pattern
versatility was limited to one direction. To address this limitation,
Guvendiren and Burdick230 reported the fabrication of strain-
responsive buckling patterns on PDMS substrates that could
control the human MSC organization with a higher complexity
and versatility. In their study, multidirectional lamellar patterns
appeared and disappeared when relaxing and stretching the
substrates, respectively, in various directions and angles. They
observed that the preferential alignment of MSCs in the presence
of lamellar topography was completely eliminated by stretching
the platform, producing a disorganized cell arrangement. Such
platforms can have considerable potential for targeted disease
modeling and drug discovery.
4.4 Drug screening
Drug development and pharmacokinetics studies using animal
models are costly, time consuming and arguably unethical
when using an extensive number of animals.231 Therefore,
the early stages of drug discovery rely on toxicity testing
using in vitro cell-based assays, to at least exclude poor ther-
apeutic candidates before animal testing.94,232 To increase their
predictive accuracy, the cell-based systems are expected to
effectively represent essential aspects of in vivo conditions.
However, a substantial number of new therapeutic agents fail
in late-stage human drug testing,233 calling for better in vivo-
mimetic culture platforms.
The cell behavior is greatly regulated by the cell’s genetic
codes and its communications with neighboring cells.234 Therefore,
the spatial position of cells is crucial for their functionality235 and
their response to a therapeutic agent. For example, multicellular
tumor spheroids have been shown to provide more physiologically
relevant models for anticancer drug screening compared to
monolayer cultures, because the intercellular adhesion in
tumor spheroids can assist tumor cells in evading the cytotoxic
effects of anticancer drugs.236 Cell patterning on engineered
extracellular environments and, then replicating the cultured
cells in miniaturized regular arrays can improve cell-based drug
testing.232,237 For example, tumor-on-a-chip systems compris-
ing arrays of tumor spheroids have been presented by several
researchers to promote cancer drug discovery238 by providing a
large number of uniform tumor spheroids for enhanced drug
screening.239
This journal is © The Royal Society of Chemistry 2019

Materials Horizons
5 Summary and outlook
Cells in their biological ECM reside within a complex 3D
network, in which every physical and chemical detail is essential
for cell functionality. The oversimplified monolayer cell cultures
have appreciably promoted our understanding of cell function.
Nonetheless, they are notoriously inadequate for successful clinical
applications. Adapting to their new environment, cultured cells
lose specific properties over time and deviate from their in vivo
behaviors. Thus, there has been an increasing demand for culture
platforms that better emulate biological conditions.
A wide range of ordered and random topographical surfaces
(identified as 212D substrates), capable of better mimicking the
natural ECM, and their impact on cell behavior were detailed in
this review. With a relatively simple fabrication procedure, 212D
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platforms show great potential in advancing biomedical appli-
cations by retaining in vivo cell functionalities, such as their
proliferative capacity. This can be significantly important for
cell transplantation and tissue engineering, where the cells
should display a long-term regeneration upon reinfusion into
the body. In addition, topographical structures with tunable
characteristics that induce 3D cell morphology furnish exciting
opportunities for better understanding cellular response to
environmental cues. Stimuli-responsive platforms are another
type of novel artificial ECMs that are expected to contrive
the dynamic investigation of cellular responses. Despite these
advances, major challenges remain as innumerate factors
simultaneously contribute to cell behavior. Nonetheless, recent
advances in micro- and nanofabrication techniques have paved
the route to studying a single cell population and to address
these challenges.
Topographical substrates are also more desirable for cell
imaging and high-throughput screening in comparison with
3D models, in which optical-readouts can be particularly
cumbersome. Nonetheless, in certain applications, such as
cancer research and producing implantable tissue constructs,
spheroids and 3D porous scaffolds can be better options as they
better emulate in vivo ECMs. However, a judicious selection
between topographical surfaces and 3D models depends on the
efficiency and performance of the models to achieve a targeted
outcome as well as on sample handling, complexity, reprodu-
cibility and economical factors.
Conflicts of interest
There are no conflicts of interest to declare.
Acknowledgements
Support from Bourses d’excellence TransMedTech (to M. M.
and V. A.), FRQNT (The Fonds de recherche du Québec – Nature
et technologies to V. A.), CRC (Canada Research Chairs program
to X. B. and D. K. H.) and NSERC (Natural Sciences and
Engineering Research Council of Canada to X. B. and D. K. H.
(Discovery Grant 2017-04489)) is gratefully acknowledged.
This journal is © The Royal Society of Chemistry 2019
View Article Online
Review
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