ARTICLE IN PRESS

Biomaterials 28 (2007) 5087–5092

www.elsevier.com/locate/biomaterials

Review

Microengineered hydrogels for tissue engineering

Ali Khademhosseinia,b, Robert Langera,c,
a
Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
b
Department of Medicine, Center for Biomedical Engineering, Brigham and Women’s Hospital, Cambridge, MA 02139, USA
c
Departments of Chemical Engineering and Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
Received 8 June 2007; accepted 6 July 2007
Available online 17 August 2007

Abstract

Hydrogels have been extensively used in various biomedical applications such as drug delivery and biosensing. More recently the
ability to engineer the size and shape of biologically relevant hydrogels has generated new opportunities in addressing challenges in tissue
engineering such as vascularization, tissue architecture and cell seeding. Here, we discuss the use of microengineered hydrogels for tissue
engineering applications. We will initially provide an overview of the various approaches that can be used to synthesize hydrogels with
controlled features and will subsequently discuss the emerging applications of these hydrogels.
r 2007 Published by Elsevier Ltd.

Keywords: Microengineered hydrogels; Tissue engineering

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5087
2. Synthesis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5088
2.1. Emulsification. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5088
2.2. Photolithography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5088
2.3. Microfluidics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5089
2.4. Micromolding. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5089
3. Tissue engineering applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5090
3.1. Top-down tissue engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5090
3.2. Bottom-up tissue engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5090
4. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5091
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5091
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5092

1. Introduction principles of engineering, physical sciences and medicine

[1]. In most cases, cells are seeded on biodegradable
Tissue engineering is an approach in generating a materials that have been fabricated in the form of porous
renewable source of transplantable tissues by using the scaffolds. As the cells deposit their own extracellular matrix
Corresponding author. Harvard-MIT Division of Health Sciences and
and as the material is degraded a tissue-like structure is
formed that can be subsequently transplanted. Despite
Technology, Massachusetts Institute of Technology, Cambridge, MA
02139, USA.
enormous advances in tissue engineering, which include the
E-mail addresses: alik@mit.edu (A. Khademhosseini), clinical approval of tissue engineered skin, a number of
rlanger@mit.edu (R. Langer). scientific barriers still prevent the fabrication of more

0142-9612/$ - see front matter r 2007 Published by Elsevier Ltd.

doi:10.1016/j.biomaterials.2007.07.021
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5088 A. Khademhosseini, R. Langer / Biomaterials 28 (2007) 5087–5092

complex organs. These challenges include the difficulties in vascularization, cell seeding and tissue complexity within
generating vascularized tissues as well as in mimicking tissue engineered constructs.
the complex structure and architecture of biological
tissues. 2. Synthesis
In the body, cells reside in close proximity to blood
vessels that supply tissues with nutrients and oxygen and To generate microengineered hydrogels the crosslinking
remove waste products and carbon dioxide. Therefore, to process must be controlled with a high degree of spatial
engineer functional tissues it is important to engineer a resolution. A number of techniques have been developed
vascular network that can perform a similar function. In that can be used to generate hydrogels with dimensions
one approach scaffolds are seeded with endothelial and as small as a few tens of nanometers. These techniques
smooth muscle cells and/or induced to release growth can be categorized into one of the following categories:
factors that promote angiogenesis into the engineered emulsification, photolithography, microfluidic synthesis
scaffold. The major challenge in this approach is that and micromolding.
during the time required to generate proper vascularization
many cells may starve for oxygen and nutrients and lose 2.1. Emulsification
their viability. Another approach to minimize this chal-
lenge is to pre-vascularize the engineered tissues prior to Emulsification is the most widely used method for
implantation by generating artificial microvasculature fabricating microgels. In this process a multi-phase mixture
within the scaffolds that can integrate with the host’s is stirred to generate small aqueous droplets of the
vasculature upon implantation. The major challenges in hydrogel precursors within an organic phase. The size of
this approach are the difficulty in fabricating such the droplets can be controlled by the degree of mechanical
controlled structures as well as ensuring their proper agitation, viscosity of each phase, as well as the presence of
function after implantation. surfactants that can modify the surface tension between the
Cell–cell contact and tissue architecture are other two phases. The resulting droplets can be gelled using a
important parameters that regulate cell behavior. For variety of crosslinking mechanisms to generate spherical
example, in the bone marrow hematopoietic progenitors microgels (Fig. 1A). This process can be used to fabricate
and stem cells closely interact with osteoblasts and microgels made from a variety of materials including
endothelial cells [2]. Although, cells within tissue engi- agarose, alginate and collagen. By adding cells to the
neered scaffolds have a certain capacity to ‘self-assemble’ aqueous phase, cell-laden microgels can be fabricated for
to regain important aspects of their cell–cell interactions, applications such as immunoisolation, as carriers within
many of these interactions are permanently lost during bioreactors or for analyzing stem cell biology [5].
tissue isolation and seeding processes. Furthermore, it is The major advantage of emulsification is the ease with
typically difficult to obtain uniform cell seeding throughout which it can be used to generate microgels. Depending on
the scaffolds, with most cells seeding in the periphery of the the process conditions the size distribution in the gels
scaffolds. Therefore, the ability to control cell–cell inter- can be minimized, however, the process typically has a
actions and proper tissue architecture as well as uniform larger size distribution than other synthesis approaches.
cell seeding may aid in generation of functional tissue Furthermore, there is little control of the resulting
constructs. shapes since the emulsification process typically produces
Microengineered hydrogels (i.e. hydrogels with features spherical droplets.
that are in the order of a few microns in at least one
dimension) are potentially powerful engineering tools to 2.2. Photolithography
overcome a number of tissue engineering challenges [3].
The use of microscale hydrogels in tissue engineering dates Photolithography is a technique that has been widely
back to some of the earliest attempts at generating developed for the microelectronics industry. More recently,
transplantable tissues. For example hydrogel microcap- photolithographic techniques have been used for a variety
sules were used in cell microencapsulation, a process in of biomedical applications to engineer microengineered
which transplanted cells were protected from the host’s hydrogels. This has been partly enabled by the develop-
immune system by being immobilized within a semi- ment of synthetic and natural photocrosslinkable pre-
permeable membrane [4]. However, more recently the polymers that can crosslink to form hydrogels [3]. In
ability to engineer the properties of hydrogel materials such photolithographic processes a thin film of a polymer is
as adhesiveness, stiffness, cell signaling potential, size and exposed to UV light through a mask. As the light reaches
shape have enabled a wide range of new applications in the photosensitive polymer through the transparent regions
tissue engineering. Here we will briefly discuss the synthesis of a mask it causes a photoreaction that crosslinks the
and the emerging applications of microscale hydrogels for polymer (Fig. 1B).
tissue engineering. We will first discuss the approaches used Photolithography can be used to create microstructured
to fabricate hydrogels with microscale features and then hydrogel scaffolds or to immobilize cells within microengi-
discuss their applications in addressing the challenges of neered hydrogels. A number of studies have demonstrated
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unique flow properties within microfluidic channels and

the precision with which these devices can be fabricated
present a unique opportunity in generating hydrogels with
controlled features. A common method of using micro-
fluidic systems to generate microparticles is by using
multiphase systems. In these approaches the viscous and
surface tension forces are used to create homogeneous
particles that can be crosslinked to form microscale
hydrogels (Fig. 1C). A range of particle sizes and shapes
can be created by generating the properly designed
microfluidic channels. For example, by changing the
dimensions of the microchannels, the flow rates and the
droplet shapes it is possible to create hydrogels in the form
of spheres and rods [11].
An interesting aspect of microfluidic fabrication of
microgels is that the spatial properties of hydrogels can
be controlled. For example, by using a microfluidic device
that can create concentration gradients of two or more
inlets it is possible to create hydrogels with controlled
gradients of signaling molecules or material properties
embedded in the hydrogel [12]. These hydrogels can be
used for various tissue-engineering applications in which
concentration gradients are desired in the scaffolds. In
addition, it is possible to generate Janus particles (i.e.
particles in two or more distinct sides) by flowing multiple
streams and generating droplets of the two or more regions
Fig. 1. Schematic diagram of the techniques to microengineer the size and [13]. Finally, it is possible to merge microfluidic techniques
shape of hydrogels: emulsification (A), photolithography (B), microflui- with other approaches such as photolithography [14–16].
dics (C) and micromolding (D).
For example, it is possible to flow hydrogels containing
cells within microfluidic channels and then capture the cells
within particular regions by crosslinking the surrounding
hydrogels by using a photomask [17]. Also, by using this
that encapsulated cells within photocrosslinkable microgels approach hydrogels can be fabricated with engineered
can be made from various types of synthetic polymers such bar-codes and analysis regions for biosensing and high-
as poly(ethylene glycol) (PEG) [6,7]. In addition, natural throughput applications [15].
photocrosslinkable pre-polymers have been developed to
enable the formation of microengineered hydrogels from 2.4. Micromolding
natural materials [8]. Photolithography can also be used to
conjugate chemical entities to hydrogels with controlled Micromolding is another technique that is capable of
spatial resolution [9,10]. generating hydrogels with controlled features. Micromold-
The resolutions that can be achieved by using photo- ing has become particularly appealing due to soft
lithography ranges from sub micron scale to millimeters. lithography which has enabled easy fabrication of poly
Considering that a cell is about 10 microns in diameter, (dimethyl siloxane) (PDMS) molds from prefabricated
photolithography can be used to generate structures that silicon wafers. Thus by using micromolding approaches it
are much smaller or much larger than a cell making it is possible to generate a great variety of micromolded
suitable for biological applications such as tissue engineer- structures in a simple manner. To generate micromolded
ing. Potential disadvantages with photolithography include hydrogels, precursor polymers are initially molded and
the need for photocrosslinkable materials, as well as the subsequently gelled to generate structures of a variety of
effects of UV light on cell function. Furthermore, since shapes and sizes [18–20] (Fig. 1D). In addition to PDMS,
photolithography is inherently a 2D process it forms other materials could also be used for micromolding. For
structures that may require further assembly to generate example, by using a novel fluoro-based material with
3D tissues. higher surface energies and enhanced fabrication resolution
it was possible to micromold nanoscale particles of
2.3. Microfluidics controlled shapes and sizes [21]. Although the immediate
application of such high-resolution nanoparticles is in drug
Microfluidics has emerged as a potentially powerful delivery, it is envisioned that the ability to engineer the
method for generating microengineering hydrogels. The nanoscale topography of hydrogels will be important in
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generating improved tissue engineering scaffolds. Micro- proximity to the engineered hydrogel microchannels
molding has also been used to generate microengineered maintain their viability while cells that are further from
hydrogels from a variety of materials including hyaluronic these channels lose their viability over time [20]. Micro-
acid (HA) [8,19], chitosan [18] and PEG [22]. engineered hydrogel microchannels can be fabricated from
Until recently micromolding techniques were unable to various hydrogels including collagen, HA or PEG and
fabricate microengineered hydrogels of controlled shapes therefore show great potential in tissue engineering
and sizes from a class of materials that require the addition applications [29].
of gelling agents such as divalent cations. These materials Microengineered hydrogels can also be used to control
encompass a large class of hydrogels including alginate and the interactions of cells relative to each other in 3D
fibrin. To alleviate this limitation hydrogel micromolds scaffolds. For example to control the size of cell aggregates
have been developed that deliver the crosslinking agent in a in 3D, dielectrophoretic methods have been used to localize
controlled manner. Therefore, these molds enable molding cells within specific regions of hydrogels that can be
the hydrogel precursors and subsequently delivering the subsequently gelled [30]. Using these approaches it is
curing agent in a desired manner [23]. possible to probe the effects of cell–cell interactions in 3D
Finally it is possible to use micromolding approaches to and to generate tissue engineered structures with control of
generate 3D hydrogel microstructures. This can be the location of the cells relative to each other. Another
accomplished by first using a sacrificial template around approach is to generate 3D microengineered scaffolds with
which the hydrogel can be formed. This approach can be appropriate tissue architecture. These scaffolds can be
used to generate 3D interconnected macroporous hydro- fabricated using either standard printing technologies or by
gels in which the macromers were formed around a packed using some of the techniques that were previously descri-
bed of polymeric beads that were subsequently dissolved bed. For example, it is possible to fabricate layers of hydro-
[24]. Also, to generate microfluidic channels within hydro- gels (either alone or containing cells) with desired porosity
gels a dissolvable gelatin-based template was used [25]. and structure. These layers can be stacked on top of each
other or used as is to generate 3D scaffolds with desired
3. Tissue engineering applications architectures. For example, a layer-by-layer microfluidic
technique was used to immobilize cell-matrix assemblies to
Microfabrication techniques are potentially powerful build multilayer constructs that mimicked the arterial
tools in tissue engineering since they can be used to structure by using three types of vascular cells [31].
replicate structures that are in the order of 0.1–10 mm, to
control the microenvironment of individual cells, 10- 3.2. Bottom-up tissue engineering
400 mm to control the structure of clusters of cells as
well as 4400 mm to control the interactions between Tissue engineered constructs can also be fabricated by
multiple cell clusters. Thus the merger of microengineered the assembly of smaller building blocks. This approach
hydrogels and microfabrication techniques has significant mimics much of the native biology that is often made from
potential to generate tissue constructs that can overcome repeating functional units. For example, in the liver, the
the limitations associated with the current tissue engineered sinusoid is the repeating functional unit. Bottom-up
constructs. Recently, two different approaches have approaches can be used to generate functional units that
emerged in using microengineered hydrogels for tissue can be assembled in a modular approach to generate larger
engineering that can be classified as either ‘‘top-down’’ or scaffolds. An interesting example of the use of modular
‘‘bottom-up’’. components for generating tissues was recently demon-
strated [32]. In this approach rod-shaped collagen micro-
3.1. Top-down tissue engineering gels that were seeded with HepG2 hepatocytes on the inside
and endothelial cells on their surfaces were ‘packed’
Top-down tissue engineering approach utilizes micro- together within a bioreactor and perfused with medium
engineering approaches to control the microscale features or whole blood (Fig. 2A and B). It was demonstrated that
of relatively large pieces of hydrogels. Early examples of the spaces between the modules formed interconnected
the top-down tissue engineering approaches have aimed to channels that exhibited delayed clotting times. In another
generate controlled featured within existing tissue engineer- approach, shape controlled cell-laden microgels were
ing scaffolds. For example, to engineer microvasculature created by micromolding photocrosslinkable hydrogels
within tissue engineering scaffolds a number of approaches such as HA. The rectangular microgels generated using
have been used to design the shapes of the scaffolds and this approach could be seeded with different cell types and
then to micromold these structures from biomaterials assembled to generate 3D tissue structures made from
[26,27]. More recently the ability to engineer the shapes various cell types with controlled architecture and cell–cell
and sizes of hydrogels has enabled the engineering of interactions. The gels could be subsequently further cross-
hydrogel-based microfluidic channels that can be poten- linked into each other to stabilize their interactions.
tially used to engineer tissue microvasculature [28]. It has Tissue printing is an emerging approach that can also be
been demonstrated that cells that are seeded in close used to form tissues from smaller building blocks [33]. By
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A. Khademhosseini, R. Langer / Biomaterials 28 (2007) 5087–5092 5091

Fig. 2. Bottom-up tissue engineering by using microengineered hydrogels. A schematic of a cell-laden microengineered hydrogels (top) and a packed bed
of hydrogels (bottom) (A). Cells can be encapsulated within microgels of controlled shapes (B). The modules can be ‘assembled’ by a number of
mechanisms. For example, spherical microgels can be fit in larger microgels using a lock-and-key process (C).

using tissue printing it is possible to generate microvascu- Also, it is envisioned that the ability to engineer functional
lature and desired architecture in tissues. Although a tissue units can be used to generate a ‘modular approach’
number of challenges such as ejector clogging and poor to tissue engineering in which smaller building blocks can
tissue mechanics have limited the current applications of be assembled to generate larger tissues with appropriate
this technology, it is anticipated that through further function and structure. Overall, it appears that our ability
research tissue printing will become a powerful ‘bottom-up’ to engineer microgels and microscale hydrogels has
approach to engineer complex 3D tissues. significant potential in overcoming many of the challenges
that have plagued the field of tissue engineering.

4. Conclusion
Acknowledgments
We have described the use of microengineered hydrogels
in tissue engineering. Over the past few years the ability to The authors would like to acknowledge the financial
engineer the shapes and sizes of biologically relevant support from the National Institute of Health, the Draper
hydrogels has led to new approaches in generating tissues. Laboratories and the Coulter Foundation. We also would
These approaches can be used to engineer microvasculature like to thank Alborz Mahdavi, Mark Brigham and Amir
and tissue architecture inside cell-containing hydrogels. Manbachi for helpful discussions.
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5092 A. Khademhosseini, R. Langer / Biomaterials 28 (2007) 5087–5092

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