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Biotechnol. J. 2014, 9, 16–27 DOI 10.1002/biot.201300187
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1 Introduction
Human physiology and pathophysiology require understanding
of cells and tissues, their interactions, and how
they generate organ-level functions. Because of the limitation
in directly observing and manipulating the human
body and underlying cell and tissue structures in vivo,
experimental studies of human physiology have relied on
ex vivo biological models, such as purified biomolecules,
cultured cells, and model organisms [1–4]. As the physiological
relevance of such models with respect to humans
increases, the experimental complexity, along with
required time, cost, and resources, also increases. Medical
and life science researchers have thus adopted models
that are as simple, robust, and reproducible as possible
but still sufficiently represent the physiological phenomena
of interest [5–8].
Cell cultures are often the ex vivo models of choice.
However, conventional 2D and 3D static cell culture models
often fail to reproduce the critical aspects of human
physiology, because cell culture approaches can be difficult
to adapt dynamic 3D microenvironments and the
simultaneous study of multiple tissues and their interactions
[5, 8–10]. For example, 3D cell culture models, in
Review
Physiologically relevant organs on chips
Kyungsuk Yum1,2, Soon Gweon Hong1, Kevin E. Healy1 and Luke P. Lee1
1 Department of Bioengineering, University of California, Berkeley, CA, USA
2 Department of Materials Science and Engineering, University of Texas, Arlington, TX, USA
Recent advances in integrating microengineering and tissue engineering have generated promising
microengineered physiological models for experimental medicine and pharmaceutical
research. Here we review the recent development of microengineered physiological systems, or
also known as "ogans-on-chips", that reconstitute the physiologically critical features of specific
human tissues and organs and their interactions. This technology uses microengineering
approaches to construct organ-specific microenvironments, reconstituting tissue structures, tissue–tissue
interactions and interfaces, and dynamic mechanical and biochemical stimuli found in
specific organs, to direct cells to assemble into functional tissues. We first discuss microengineering
approaches to reproduce the key elements of physiologically important, dynamic mechanical
microenvironments, biochemical microenvironments, and microarchitectures of specific tissues
and organs in microfluidic cell culture systems. This is followed by examples of microengineered
individual organ models that incorporate the key elements of physiological microenvironments
into single microfluidic cell culture systems to reproduce organ-level functions. Finally,
microengineered multiple organ systems that simulate multiple organ interactions to better represent
human physiology, including human responses to drugs, is covered in this review. This
emerging organs-on-chips technology has the potential to become an alternative to 2D and 3D cell
culture and animal models for experimental medicine, human disease modeling, drug development,
and toxicology.
Keywords: Microengineering · Microfluidics · Organs-on-chips · Physiologically relevant microenvironment · Tissue engineering
Correspondence: Prof. Luke P. Lee, Department of Bioengineering,
408C Stanley Hall, Berkeley, CA 94720-3220, USA.
E-mail: lplee@berkeley.edu
Abbreviations: 3D, three-dimensional; 2D, two-dimensional; μCCA, microscale
cell culture analog; ADME, absorption, metabolism, distribution, and
elimination; AVP, arginine vasopressin; CCA, cell culture analog; ECM,
extracellular matrix; HUVEC, human umbilical vein endothelial cell; IdMOC,
integrated discrete multiple organ co-culture; IMCD, inner medullary collecting
duct; PBPK–PD, physiologically based pharmacokinetic–pharmaco-
dynamic
Received 24 APR 2013
Revised 16 SEP 2013
Accepted 28 OCT 2013
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which cells are grown within 3D scaffolds, allow cells to
interact with neighboring cells and the extracellular
matrix (ECM) [11]; such cell–cell and cell–ECM interactions
improve tissue-specific functions. However, 3D cell
culture models do not reconstitute highly dynamic
microenvironments of living organs crucial for reproducing
organ-specific functions, such as dynamic mechanical
microenvironments, time-varying gradients of biomolecules,
and tissue–tissue interfaces. Therefore, despite
their experimental complexity, lack of experimental
throughput, and cost, animal models continue to be used
[2, 6, 12]. However, in addition to ethical concerns, the relevance
of animal models to human physiology is often
questionable as data obtained from animals can prove difficult
to extrapolate to humans [2, 6, 9, 10, 12].
The integration of microengineering and tissue engineering
has recently introduced a new biological model
that has the advantages of both in vitro cell culture and in
vivo animal models, namely simplicity, high-throughput,
and physiological relevance [3, 5]. For example, microfabrication
techniques, such as replica molding and microcontact
printing, can create microscale structures and
patterns that can be designed to construct physiologically
relevant mechanical, biochemical, and structural microenvironments
[13, 14]. In particular, microfluidics, the science
and technology that manipulate small amounts of
fluids in channels with dimensions of tens to hundreds of
micrometers, is inherently ideal for such applications [15].
Microfluidics offers the ability to precisely control fluid
flows for transporting nutrients, generating biomolecular
gradients, and applying a flow-induced shear stress and
mechanical strain to cultured cells [4].
The early applications of microengineering and
microfluidics to cell biology emerged from surface engineering
of 2D cellular microenvironments to control the
shape, location, and growth of cells, cell–cell interactions,
and the expression of tissue-specific functions of cells [3,
13, 14, 16–18]. This technology has also enabled cellseeded
3D scaffolds with microfluidic vascular networks
[19]. As the technology matures, recent efforts have
moved toward creating physiologically relevant microenvironments
for specific tissues and organs [5, 9, 10, 12].
This emerging technology, named organs on chips,
uses microfabrication techniques to construct organ-specific
cell culture microenvironments that reconstitute tissue
structures, tissue–tissue interactions and interfaces,
and dynamic mechanical and biochemical stimuli found
in specific human organs to create functional tissue and
organ models. For example, organ-specific 3D microarchitectures,
microfluidic vascular networks, biochemical
gradients, and mechanical stimuli have been incorporated
into single microfluidic cell culture systems. Because
such physiological complexities are introduced by engineering
the microenvironment, this approach maintains
the simplicity and throughput of cell culture models [5].
Furthermore, because this approach can use human cells
and culture them in microenvironments that mimic those
in the human body, the organs-on-chips has the potential
to better represent human physiology than animal models.
Figure 1 shows representative microengineered physiological
systems developed in the past decade, including
a microfabricated array bioreactor for 3D liver culture with
cross-flow perfusion [20, 21], a microscale cell culture analog
(CCA), a microscale physical representation of a physiologically
based pharmacokinetic (PBPK) model for toxicology
and drug development [22, 23], a dynamic cell culture
array with continuous perfusion of medium [24], a
liver-on-a-chip device based on the dynamic perfusion
cell culture [25], and a mechanically active lung-on-a-chip
device that reconstitutes multiple physiological features
of the human lung [26].
Here we review the recent development of microengineered
physiological (microphysiological) systems, or
organs on chips, that reproduce the physiologically relevant,
critical features of specific organs and organ–organ
interactions in the human body. We first review microengineering
approaches to construct the key elements of
physiologically important, dynamic mechanical and biochemical
microenvironments and 3D microarchitectures
of human organs in microfluidic devices. We then give
Figure 1. Development of microengineered physiological model systems: microfabricated array bioreactor for 3D liver culture with cross-flow perfusion
(2001) [21], microscale cell culture analog (CCA) (2004) [3], dynamic cell culture system with continuous perfusion (2005) [24], liver-on-a-chip device with
endothelium-like barriers (2007) [25], and mechanically active lung-on-a-chip device (2010) [26]. Reproduced from [3, 21, 24-26] with permission.
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examples of individual microengineered organ models
that incorporate such key elements of microengineered
microenvironments into single microfluidic cell culture
systems to reproduce organ-level functions of specific
organs in vitro. We finally discuss multiple organ model
systems that simulate multiple organ interactions to better
reproduce human physiology, particularly for predicting
human responses to drugs for drug development.
2 Key elements of microenvironments
Microengineered cell culture systems have created various
dynamic microenvironments found in the human
body, including blood flow, fluid-induced shear stress, and
gradients of oxygen, growth factors, and other biochemical
signal (Fig. 2). For example, Leclerc et al. [27–29] developed
a microfluidic device with continuous fluid perfusion
to supply the nutritional medium for cell culture and
studied the effects of perfusion flow rate and glucose and
oxygen supply on the cell culture (Fig. 2A). Compared to
diffusion-based nutrient transport in traditional static cultures
in dishes, the perfusion cell culture enhanced the
nutrient transport via convection through the microfluidic
medium channels. The perfusion device also led to significant
increase of albumin production of fetal human hepatocytes
compared to static culture conditions [28]. Hattori
et al. [30] also developed a microfluidic perfusion cell culture
array device, “microenvironment array chip,” in
which they created combinatorial cell culture microenvironments
composed of four types of soluble factors and
ECMs (total 16 different microenvironments) for screening
cell culture environments (Fig.  2B). Similarly, King
et al. [31] demonstrated a microfluidic parallel perfusion
cell culture system that can control the dynamics of soluble
cellular microenvironments by using a “flow-encoded
switching” design strategy. The “flow-encoded switching”
strategy uses a laminar flow microfluidic device to
control the temporal aspects of cellular stimuli (state of the
network) with the ratio of two flow rates (single flow control
parameter).
Recent microengineered cell culture systems have
been focused on reproducing physiologically relevant
dynamic microenvironments of specific tissues and
organs. For instance, Maidhof et al. [32] developed a bioreactor
that simultaneously provides two critical factors
for the development of cardiac tissues: synchronous
medium perfusion and tissue contraction driven by electrical
stimulation. The simultaneous application of nutrient
perfusion and electrical stimulation improved the differentiation
of cardiac cells and their assembly into functional
cardiac tissue constructs.
In this section, we will discuss how we can use microengineering
approaches to construct the key elements of
physiologically relevant, dynamic mechanical microenvironments,
biochemical microenvironments, and microarchitectures
of specific organs and tissue in microfluidic
devices.
2.1 Dynamic mechanical microenvironments
Cells and tissues in living organs experience various
mechanical forces. For example, endothelial cells that line
the interior surface of blood vessels are exposed to a fluidinduced
shear stress from blood flow, shear stress across
the vessel from interstitial plasma flow, and the interfacial
mechanical force between the cells and the surrounding
matrix [33, 34]. Such mechanical stimuli have been recognized
as important factors for various physiological
processes and critical determinants of differentiated
functions of cells and tissues [33, 34].
In this section, we will discuss dynamic mechanical
microenvironments: flow-induced shear stress and
dynamic mechanical strain.
2.1.1 Flow-induced shear stress
Early microfluidic cell culture systems developed to generate
flow-induced shear stress have mostly been used to
study the effects of fluid-induced shear stress on cell
adhesion, mechanics, morphology, and growth [35–39].
Recent studies have focused on reproducing physiologically
relevant shear stresses to understand their effects in
the context of specific tissues and organs [40]. For example,
Huh et al. [41] constructed a microfluidic airway model
of the human lung that consists of two microfluidic
channels, which represent apical (airway lumen) and
basal compartments of the airway epithelium, respectively,
and are separated by a porous polyester membrane.
They used this device to reproduce three physiological
conditions by introducing air, single-phase liquid, and
liquid plugs into the microchannel: (i) normal breathing,
(ii) the motion of liquid during total liquid ventilation or
fetal breathing movements in the developing lung, and
(iii) lung injury during airway reopening, respectively.
This lung-on-a-chip device revealed that the fluid
mechanical stresses generated by the propagation and
rupture of the liquid plug can induce significant injury of
the small airway epithelial cells. Interestingly, the device
also generated cracking sounds when plugs ruptured and
caused mechanical cell damage.
Another example is a kidney-on-a-chip device that
reproduces luminal fluid shear stress (0.2–20  dyn/cm2)
and transepithelial osmotic gradients produced by urinary
flow in the collecting duct system of the kidney [42,
43]. Jang et al. [42, 43] used the kidney-on-a-chip device
to study the role of luminal fluid shear stress in the reorganization
of actin cytoskeleton and the translocation of
water transport proteins (aquaporin-2) of inner medullary
collecting duct (IMCD) cells of the kidney.
Microfluidic cell culture systems were also designed
to study how fluid forces modulate angiogenesis. To
understand the collective effects of fluid and chemical
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factors on endothelial sprouting, Song and Munn [34]
developed a microfluidic cell culture system that comprises
two parallel microfluidic channels, lined with
human umbilical vein endothelial cells (HUVECs), and a
central microchannel of a 3D collagen ECM that separates
the two parallel microfluidic channels, into which
Figure 2. Key elements of physiologically relevant microenvironments. (A) Microfluidic device with continuous perfusion to supply the nutritional medium
for cell culture [28]. (B) Microenvironment array chip composed of soluble factors and extracellular matrices for screening cell culture microenvironments, in
which Chinese hamster ovary (CHO)-K1 cells were cultivated for demonstration [30]. (C) Schematic of human MSC migration in response to concentration
gradient generated by endothelial cells (EC) (top), cell migration vectors generated from phase contrast cell images for MSC-only and MSC-EC coculture conditions,
and overlays of the measured cell vectors and modeled vector fields for the two culture conditions (bottom). The insets show the correlation of two
vector fields in the outlined regions [66]. (D) Brain-on-a-chip: compartmentalized microfluidic device with microgrooves that connect the two rectangular
compartments containing two independent populations of neurons and guide the growth of dendrites and axons to form synapses in the microgrooves.
Neurons on the left expressed GFP (green fluorescent protein) whereas neurons on the right expressed RFP (red fluorescent protein). Scale bar, 150 μm [78].
(E) Computational fluid dynamic model for predicting hematocrit distribution within an engineered vascular network that mimics capillary vasculature (left),
microfabricated master mold for polymer casting of an engineered microvascular network (middle), and bilayer hepatocyte culture device that consists of an
engineered microvascular network layer for blood flow and oxygenation and a chamber for hepatocyte culture, and an nanoporous membrane that separate
the microvascular network and the hepatocyte culture (right) [84]. Reproduced from [28, 30, 66, 78, 84] with permission.
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HUVECs can migrate. This microfluidic system generated
multiple mechanical and chemical signals to recreate
the physiological microenvironment of endothelial cell
angiogenesis: tangential fluid shear stress from blood
flow, transverse interstitial shear stress, and gradients of
vascular endothelial cell growth factors. An interesting
finding is that tangential shear stress, exerted by blood
flow in vivo, attenuates endothelial cell sprouting and
transverse interstitial shear stress (e.g. stress produced
by extravasating plasma) enhances the rate of morphogenesis
and sprout formation.
2.1.2 Dynamic mechanical strain
In addition to flow-induced shear stress, cells and tissues
in the human body continuously experience organ-specific
tensile and compressive forces during the normal
operation of organs. To reproduce such mechanical
microenvironments of the human lung in vitro, Douville et
al. [44] developed a multilayered microfluidic lung-on-achip
device that uniquely mimics the combined solid and
fluid (surface-tension) mechanical stresses induced by
the cyclic wall stretching and the propagation of an
air–liquid meniscus in alveoli of the human lung. Previously
reported in vitro models of ventilator-induced lung
injury generated either cyclic stretching [45, 46] or air–
liquid interface flow over the cells on nonstretching
substrates [41, 47, 48]. This lung-on-a-chip device [44]
created more physiologically relevant mechanical microenvironments
for alveolar epithelial cells during ventilation
by simulating both fluid and solid mechanical stresses.
This study showed that combined solid and fluid
mechanical stresses (cyclic stretch and surface tension
forces, respectively) significantly increase cell death and
detachment compared to solid mechanical stress alone,
supporting clinical observations that cyclic stretch alone
is not sufficient to induce the level of the cell injury as
seen in ventilator-induced lung injury.
Another example is a human-breathing lung-on-achip
device that reconstitutes mechanically active
microenvironment of the alveolar–capillary interface of
the human lung [26, 49]. Fluid-induced shear stress was
generated by introducing the culture medium into the
capillary channel, which creates a physiological level of
fluid-induced shear stress (1 dyne/cm2). Introduction of
air into the alveolar channel created air-induced shear
stress. In addition to these stresses, the lung-on-a-chip
device reproduced the cyclic strain from the breathing
movement in the human lung (10% at 0.2 Hz) by applying
cyclic suction to the hollow side chambers, thus causing
the mechanical stretching of the flexible membrane
between the alveolar and capillary compartments [26, 49].
The lung-on-a-chip device was also used to create a
human disease model-on-a-chip of pulmonary edema
[49].
In addition to reproducing, the mechanical microenvironments
of muscular organs, Grosberg et al. [50]
designed a heart-on-a-chip device that uses muscular
thin films (MTF), elastic biohybrid constructs that consist
of 2D engineered muscle tissues on elastomeric thin films,
to measure contractility of engineered cardiac tissues.
They also adapted the MTF-based heart-on-a-chip to
develop muscle-on-a-chip devices with both striated and
smooth muscle cells [51].
2.2 Biochemical microenvironments
Elucidating the fundamental mechanism of gradientdriven
biochemical signaling has offered new insights
into various physiological processes, including immune
responses, wound healing, cancer metastasis, and stem
cell differentiation [52, 53]. Diffusive mixing in streams of
laminar flow in microchannels at low Reynolds number
conditions leads to generating stable, spatially and temporally
controlled gradients of soluble molecules, difficult
to achieve with conventional methods [53, 54]. Early studies
with microfluidic gradient generators focused on creating
different types of biomolecular gradients and understanding
their biological effects in 2D cellular microenvironments
(i.e. chemotaxis) [55, 56]. Recent work has created
3D biochemical microenvironments that mimic
biological processes occurred in the human body [57–62].
For example, a microfluidic cell culture system that comprises
hydrogel-incorporating chambers between surface-accessible
microchannels has been used to study
angiogenesis under well-controlled gradients of growth
factors in 3D microenvironments [61–64].
Another approach to generate physiologically relevant
biochemical gradients is to pattern chemoattractantsecreting
(source) and chemoattractant-scavenging
(sink) cells in defined locations in microfluidic channels.
For instance, Torisawa et al. [65] developed a microfluidic
cell culture system that recapitulates physiological gradients
of chemokine (CXCL12) in cancer-stroma microenvironments
by patterning chemokine-secreting cells
(source), chemokine-scavenging cells (sink), and migrating
cancer cells in spatially defined positions inside
microchannels. This approach enabled efficient chemotaxis
under shallower yet more physiological gradients of
chemoattractants and showed that the presence and
location of sink cells is critical for efficient chemotaxis.
Similarly, Eng et al. [66] developed a shape-coded hydrogel-based
method to create patterned 3D cellular
microenvironments and used this method to control the
geometrical pattern of the coculture of human mesenchymal
stem cells (MSC) and endothelial cells (EC) (Fig. 2C).
They used this system to study the migration of MSC in
the controlled biochemical gradient generated by the patterned
coculture.
Other examples include a perfusion bioreactor in a liver
model with co-cultures of hepatocytes and fibroblasts,
which generates physiological oxygen gradients [67].
A perfusion-based microfluidic system was also used to
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study the dynamic motion of primary human hepatocytes
cells in 3D cell culture environments [68]. A paper-based
multi-layer cell culture system (cells-in-gels-in-paper) was
also developed to study the behavior of tumor cells and
microvascular endothelium in response to oxygen gradients
[69, 70].
2.3 Physiologically relevant microarchitectures
Recent advances in microfabrication have brought more
complex and sophisticated cell culture microenvironments
that reconstitute in vivo-like 3D microarchitectures,
including multiple tissue structures, tissue–tissue
interfaces, and microvascular networks [5]. In this section,
we will discuss three major approaches to construct
the physiologically relevant microarchitectures: microstructures
in single-layer microfluidic devices, 3D compartmentalization,
and microfluidic vascular networks.
2.3.1 Microarchitectures in single-layer microfluidic
devices
Fabricating microstructures and culturing different types
of cells in predefined regions can reproduce the structural
microenvironment in the human body, critical to generate
functional tissue and organ models [34, 61–65, 71].
For example, Sudo et al. [60] developed a microfluidic
coculture system that consists of an intervening 3D gel
scaffold (e.g. type I collagen) between two parallel microfluidic
channels, in which liver and vascular cells were
cultured on each sidewall of the scaffold. They used this
Figure 3. Microengineered individual organ models. (A) Liver-on-a-chip: a biologically inspired artificial liver model with endothelium-like barriers that mimic
the endothelium of the sinusoid in the human liver. Scale bar, 50 μm [25]. (B) Multi-layer microfluidic kidney-on-a-chip. Kidney tubular epithelial cells cultured
on the membrane are exposed to a flow-induced shear stress and a transepithelial osmotic gradient defined by the simulated urinary flow in the
microchannel. The images below show the effects of luminal fluid shear stress and arginine vasopressin (AVP) stimulation on F-actin (red) and water transport
proteins (green), and x–z reconstruction under the images. Scale bar, 10 μm [43]. (C) Biologically inspired human breathing lung-on-a-chip device. The
lung-on-a-chip device reproduces physiological breathing movements in the living lung by mechanically stretching the membrane that mimics the alveolarcapillary
interface [49]. (D) Endothelialized microfluidic vascular networks in engineered 3D tissues. Schematic cross-sectional view of a section of microfluidic
vessel networks illustrating (i) microvessel formation, (ii) endothelial sprouting, (iii) perivascular interaction, and (iv) whole blood interaction (top left).
Schematic of microfluidic collagen scaffolds with microfluidic endothelial vessel networks (top right). Horizontal confocal images of endothelialized
microfluidic vessels (i) and views of corner (ii) and branching sections (iii) (below). Red, CD31; blue, nuclei. Scale bar: 100 μm [87]. Reproduced from
[25, 43, 49, 87] with permission.
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microfluidic coculture system to study angiogenesis in 3D
cultures of hepatocytes and microvascular endothelial
cells. A similar microfluidic device with three flow channels
and two collagen scaffolds that separate the flow
channels was also constructed to study capillary growth
and endothelial cell migration under various coculture
conditions [72].
Microfluidic coculture systems can create in vitro 3D
models for cell–cell signaling studies during disease
development and progression. For example, a simple
Y-shaped microfluidic coculture system was used to
study the transition of ductal carcinoma in situ to invasive
ductal carcinoma in breast cancer progression, where the
laminar flow-based patterning generated two side-byside
compartments to culture mammary epithelia cells
and human mammary fibroblasts in each compartment
[73]. Grafton et al. [74] developed a simple microfluidic
breast ductal system with branched microchannels of
decreasing size. They used this microfluidic system with
branched microchannels as an in vitro testing platform to
characterize targeting and toxicity of superparamagnetic
submicron particles and their use for therapeutics.
Compartmentalized microfluidic devices have also
been used to arrange neuronal cells and direct their
growth to induce the physiological connections of neurons
as found in vivo [75–79]. For example, Taylor et al. [75,
78] developed compartmentalized microfluidic devices to
guide the growth of axons and dendrites by using parallel
microgrooves, which also allowed them to visualize and
manipulate synapses and presynaptic and postsynaptic
cell bodies (Fig. 2D). Peyrin et al. [80] fabricated a similar
microfluidic system with two compartments connected
by asymmetrical microchannels, “axon diodes,” to generate
oriented neuronal networks.
2.3.2 3D compartmentalization
The concept of 3D compartmentalization that creates
microengineered compartments with physiologically
defined microenvironments and physiological interfaces
between the compartments can create 3D microarchitectures
in human organs (examples are also shown in Fig. 3)
[81]. For example, the concept of 3D compartmentalization
was applied to kidney-on-a-chip (Fig. 3B) [42, 43] and
lung-on-a-chip devices (Fig. 3C) [26, 41, 44, 49] by constructing
multi-layer microfluidic devices. A similar twolayer
microfluidic device with a porous membrane was
also designed to produce a 3D metastatic cancer model to
study the interactions between circulating breast cancer
cells and microvascular endothelium under physiological
flow conditions [82].
2.3.3 Microfluidic vascular networks
Human organs require dense microvasculature to maintain
the function of the cells in the organs. The development
of artificial microvascular networks to transport
nutrients and oxygen and remove wastes is critical not
only for developing engineered tissues for clinical applications
but also for maintaining vital functions of cells in
organs-on-chips devices; vascular networks are particularly
important for microengineered physiological systems
of highly metabolic organs, such as heart, liver, and
kidney. In addition, angiogenesis is critical for understanding
various physiological processes, including
wound healing and tumor growth [83]. Artificial microfluidic
vascular networks have also been used to mimic
physiological blood flow and meet the metabolic demands
of effective transport of oxygen and nutrients and removal
of wastes (Fig. 2E) [84, 85].
Miller et al. [86] constructed patterned vascular networks
in engineered 3D tissues by using a 3D printing
method. The vascular networks could be lined with
endothelial cells and perfused with high-pressure human
blood. The perfused vascular networks also sustained the
metabolic function of primary hepatocytes in engineered
3D tissue constructs. Endothelialized microfluidic vascular
networks constructed in 3D tissue scaffolds by using
the injection molding method [19] are another example of
physiologically functioning, perfusable vascular networks
[87] (Fig. 3D).
3 Microengineered individual organ
and tissue models
A number of microengineered physiological models have
been developed to reproduce key features of specific tissues
and organs or biological processes. In this section,
we discuss the microengineered key elements of microenvironments
(discussed in the previous section) can be
incorporated into single microfluidic cell culture systems
to construct microengineered models of specific tissues
and organs. We particularly highlight microengineered
physiological models of liver, kidney, lung, and vascular
networks.
One of early examples of organs on chips is a microengineered
liver model (Fig. 3A) [25]. This liver-on-a-chip
device reconstitutes the physiological microarchitecture
of the liver, including the hepatic cord-like structure, sinusoids,
and highly permeable endothelial cell barriers that
separate hepatocytes in the cord-like structure and sinusoids.
The endothelium-like barriers were particularly
designed to reproduce the physiologically relevant diffusive
transport of nutrients and wastes between the hepatocytes
and the sinusoid through the endothelium in the
liver. A similar concept was applied to other types of liver-on-a-chip
devices for drug screening applications [57,
88, 89].
A microengineered kidney model was also developed.
Jang et al. [42, 43] reconstituted dynamic mechanical and
biochemical microenvironments of the collecting duct
system in the kidney, including luminal fluid shear stress
(0.2 to 20 dyn/cm2) and transepithelial osmotic gradients
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exerted by urinary flow on renal tubular epithelial cells
(Fig. 3B). They constructed the kidney-on-a-chip device
by stacking two compartments, an upper flow channel
and an underlying static well, and a porous membrane
that separates the two compartments and then culturing
primary rat IMCD cells on the flow channel side of the
membrane. The kidney-on-a-chip device provides an
insight into how dynamic microenvironments, such as
fluid shear stress, hormonal stimulation, and osmotic gradients,
induce depolymerization of actins and trafficking
of water transport proteins (aquaporin-2) of the kidney
cells.
Another example is lung-on-a-chip devices that
reconstitute one or several distinct features of the human
lung, including the tissue–tissue interface, physiological
breathing movements, and the air–liquid interface of the
lung [26, 41, 44, 49]. The lung-on-a-chip device (Fig. 3C)
[26, 49] particularly reconstituted the microstructure of
the alveolar–capillary interface, which consists of the
epithelial cells of the alveolus facing air, the endothelial
cells of the capillary facing blood, and the permeable
basement membrane between the two tissue layers. This
device also reproduced multiple dynamic mechanical
microenvironments found in vivo, including the breathing
movements of the alveolus, which induces the continuous
exposure of the lung tissue to mechanical stretching, the
air-induced shear stress exerted on the epithelial cells,
and the blood-induced shear stress exerted on the
endothelial cells. This lung-on-a-chip device uniquely
reproduced the complex responses of the human lung to
bacteria and inflammatory cytokines introduced into the
alveolar space.
Perfusable vascular networks were also constructed in
3D engineered tissues (Fig.  3D) [86, 87]. For example,
Zheng et al.[87] constructed endothelialized microfluidic
vascular networks within 3D tissue scaffolds and demonstrated
their biological functionality in vitro (Fig.  3D).
They fabricated the microfluidic vascular networks by
seeding HUVECs and perivascular cells into microfluidic
vascular networks constructed in 3D tissue scaffolds (type
I collagen) by using injection molding techniques [19].
Compared to previous angiogenesis models, such as lateral
endothelial sprouting into a collagen gel isolated from
two microchannels lined with HUVECs and 3D sprouting
from microbeads in a bulk gel [34], the endothelialized
microfluidic vascular networks uniquely reproduced the
key features of vascular networks, including initiation of
angiogenesis from native-like endothelialized vessels
with luminal flow and control of mechanical and chemical
microenvironment of the endothelium. The endothelialized
microfluidic vascular networks showed the formation
of appropriate endothelial morphology and barrier functions
(Fig. 3D, i), allowing to study angiogenic remodeling
(Fig.  3D, ii), interactions between endothelial cells and
perivascular cells (human brain vascular pericytes and
human umbilical arterial smooth muscle cells) (Fig. 3D,
iii), and interactions of whole blood and endothelium
under flow (Fig. 3D, iv).
Other microengineered physiological systems have
also been developed to build heart [32, 50], muscle [51],
brain [75, 76, 78, 79, 90–92], gut [93, 94], pancreatic islet
[95], eye [77], tumor [34, 96] models.
4 Microengineered multiple organ models
An early development of microphysiological systems for
multiple organ models came from the need for new model
systems for human toxicology and drug screening to
overcome the limitation of conventional cell culture models,
particularly the lack of dynamic organ–organ interactions
[1, 2]. Shuler et al. [1, 2] proposed a CCA and a
microscale CCA (μCCA) of a physiologically based a pharmacokinetic–pharmacodynamic
(PBPK–PD) model as an
alternative to computational PBPK, cell culture, and animal
models (Fig. 4A) [6, 7, 97, 98]. The microscale CCA is
a physical representation of a PBPK model, in which multiple
cell culture compartments, representing different
organs with physiological tissue-to-tissue size ratio, are
interconnected through microfluidic channels under
physiologically relevant fluid flow conditions to predict
the time-dependent absorption, metabolism, distribution,
and elimination (ADME) of drugs in the human body and
human responses to drugs. Li [99, 100] also proposed the
integrated discrete multiple organ co-culture (IdMOC)
system to overcome the shortcomings of in vitro biological
models, such as the lack of multiple organ metabolism
and interactions (Fig. 4B). Li realized the concept of multiple
organ interactions by constructing multiple small
inner wells with cells from specific organs within a large
outer well containing the overlying medium that interconnects
the physically discrete multiple organ cells in
the small inner wells. However, the simple expansion of
static cell culture platforms for multiple organ interactions
in the IdMOC system has a limitation that it does not consider
the dynamic nature of organ–organ interactions,
such as dynamic exchange of metabolites between
organs, and the circulation system in the human body,
which changes ADME [6].
A common limitation of the μCCA and the IdMOC is
that the simple representation of individual organs by
using cell cultures may not properly reproduce organ-specific
functions. The current interest is to integrate multiple
organs-on-chips devices, each of which is designed to
reproduce the key features of specific human organs (as
discussed in the previous section). Such approaches
would allow for assessing human responses to drugs on
both individual organ and multiple organ levels, including
off-target toxicity [101].
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5 Concluding remarks
The microphysiological systems, or organ on chips, have
been emerging as a physiological model for human physiology,
drug development, and toxicology. The organs-onchips
technologies reconstitute dynamic microenvironments
that reproduce the key features of specific human
tissues and organs in microfluidic cell culture systems.
Organs-on-chips devices have already begun to serve as
an alternative to 2D and 3D cell culture models for studying
biological mechanisms in the context of specific tissues
and organs [26, 49]. The greater potential of human
cell-based organs on chips is in creating human disease
models and predicting human responses to drugs and
chemicals. For example, one of the main reasons for the
failure of new drugs is that animal models often do not
predict the efficacy and toxicity of drugs in the human
body, because of considerable difference between human
and animal metabolism (e.g. liver toxicity). Therefore, the
development of organs on chips that can predict the efficacy
and toxicity of drugs in the human body, more accurately
than animal models, could improve success rates in
clinical trials and reduce the time and cost for drug development.
Another benefit is that the precise experimental
control possible in organs on chips will allow for mechanistic
studies at high temporal and spatial resolutions, at
a level difficult to be achieved with complex animal models,
thus enhancing our understanding of fundamental
mechanisms in human responses to drugs.
There exist scientific and technological challenges for
the success of organs on chips. This emerging technology
requires further scientific validation and characterization
to define their capability and limitation for practical
biomedical applications. For example, before their use for
drug screening, characterization of organs-on-chips
devices, by using drugs of which the clinical efficacy and
toxicity are well characterized, is required to validate the
capability and limitation in predicting human responses
to drugs. Another important area for further investigation
is to develop mathematical models that can correlate data
from organs on chips and in vivo experiments to extrapolate
data obtained from organs on chips to humans,
including PK–PD models [6, 7, 12].
The wider use of organs on chips, including transferring
this technology from the laboratory to clinical and
industrial applications, requires the development of userfriendly,
standardized organs on chips systems. The
design concepts toward these goals include developing
scalable, robust, and easy-to-use systems, increasing the
compatibility with existing biological techniques, such as
high throughput screening systems, developing the onchip
capability for real-time sensing and control of cells
Figure 4. Microengineered multiple organ models. (A) Microscale cell culture analog (CCA): a microscale CCA of a physiologically based pharmacokinetic–pharmacodynamic
(PBPK–PD) model of the human body to predict human responses to drugs and their metabolites [6, 7]. (B) Integrated discrete multiple
organ co-culture (IdMOC) system, consisting of multiple inner wells with cells from specific organs, representing physically discrete organs, within a
large outer well containing the overlying medium, which interconnects the physically discrete organ cells [100]. Reproduced from [6, 7, 100] with permis-
sion.
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and tissues and their surrounding microenvironments,
and standardizing designs and interfaces to potentially
develop multiple organ systems.
In addition to constructing physiologically relevant
microenvironments from the engineering side, the supply
of relevant human cells from the biology side and their use
in organs on chips will be another important factor for the
future success. Combining organs on chips and human
induced pluripotent stem cells technologies in this regard
could solve the issue of the availability of primary human
cells and improve the relevance of organs on chips to
human physiology. More importantly, this approach
would also create patient-specific, human tissue and
organ models for personalized medicine, drug screening,
and toxicology [102–106].
The authors would like to acknowledge funding by the
National Center for Advancing Translational Sciences
(NCATS) at the National Institutes of Health (NIH) (NIHNCATS
UH2NS080691).
The authors declare no conflict of interest.
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Kyungsuk Yum is Assistant Professor
in the Department of Materials Science
and Engineering at the University of
Texas, Arlington. He received his BS in
Mechanical and Aerospace Engineering
from Seoul National University and
PhD in Mechanical Science and Engineering
from the University of Illinois
at Urbana-Champaign. Before joining
the University of Texas, he worked as a postdoctoral research associate
in Chemical Engineering at the Massachusetts Institute of Technology
and in Bioengineering at the University of California, Berkeley.
His research interests are biologically inspired materials and engineering
systems, nanobiotechnology, nano-biomanufacturing, and nano-
materials.
Luke P. Lee is Arnold and Barbara Silverman
Distinguished Professor of Bioengineering
at UC Berkeley, the Director
of the Biomedical Institute of Global
Healthcare Research & Technology
(BIGHEART) and a Co-Director of the
Berkeley Sensor & Actuator Center.
He is a 2010 Ho-Am Laureate. He
received his BA in Biophysics and PhD
in Applied Science & Technology from UC Berkeley. He has more than
10 years of industrial experience in integrated optoelectronics, Superconducting
Quantum Interference Devices (SQUIDs), and biomagnetic
assays. His research interests are bionanoscience, nanomedicine for
global healthcare and personalized medicine, and Bioinspired Photonics-Optofluidics-Electronics
Technology and Science (BioPOETS).
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© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.biotechnology-journal.com
Editorial: Latest methods and advances in biotechnology
Sang Yup Lee and Alois Jungbauer
http://dx.doi.org/10.1002/biot.201300522
Editorial: Biotechnology Journal – a review of 2013 and a preview
of 2014
Judy Peng
http://dx.doi.org/10.1002/biot.201300524
Review
Physiologically relevant organs on chips
KyungsukYum, Soon Gweon Hong, Kevin E. Healy and Luke P. Lee
http://dx.doi.org/10.1002/biot.201300187
Review
Large-scale production of red blood cells from stem cells:
What are the technical challenges ahead?
Guillaume F. Rousseau, Marie-Catherine Giarratana and
Luc Douay
http://dx.doi.org/10.1002/biot.201300368
Review
Molecular farming of human cytokines and blood products
from plants: Challenges in biosynthesis and detection
of plant-produced recombinant proteins
Nicolau B. da Cunha, Giovanni R.Vianna,Thaina da Almeida Lima
and Elíbio Rech
http://dx.doi.org/10.1002/biot.201300062
Review
Biomaterial and cellular properties as examined through atomic force
microscopy, fluorescence optical microscopies and spectroscopic
techniques
Birgit Kainz, Ewa A. Oprzeska-Zingrebe and José L.Toca-Herrera
http://dx.doi.org/10.1002/biot.201300087
Review
Microbial heterogeneity affects bioprocess robustness:
Dynamic single-cell analysis contributes to understanding
of microbial populations
Frank Delvigne and Philippe Goffin
http://dx.doi.org/10.1002/biot.201300119
Review
Algal biomass conversion to bioethanol – a step-by-step assessment
Razif Harun, Jason W. S.Yip, Selvakumar Thiruvenkadam, Wan A.
W. A. K. Ghani,Tamara Cherrington and Michael K. Danquah
http://dx.doi.org/10.1002/biot.201200353
Research Article
Recovery of Chinese hamster ovary host cell proteins for proteomic
analysis
Kristin N.Valente, Amy K. Schaefer, Hannah R. Kempton,
Abraham M. Lenhoff and Kelvin H. Lee
http://dx.doi.org/10.1002/biot.201300190
Research Article
Highly sialylated recombinant human erythropoietin production in
large-scale perfusion bioreactor utilizing CHO-gmt4 (JW152) with
restored GnT I function
John S.Y. Goh,Yingwei Liu, Haifeng Liu, Kah Fai Chan,
Corrine Wan, Gavin Teo, Xiangshan Zhou, Fusheng Xie,
Peiqing Zhang,Yuanxing Zhang, Zhiwei Song
http://dx.doi.org/10.1002/biot.201300301
Research Article
Secretory ranalexin produced in recombinant Pichia pastoris exhibits
additive or synergistic bactericidal activity when used in combination
with polymyxin B or linezolid against multi-drug resistant bacteria
Rasha Abou Aleinein, Holger Schäfer and Michael Wink
http://dx.doi.org/10.1002/biot.201300282
Research Article
Engineering stress tolerance of Escherichia coli by stress-induced
mutagenesis (SIM)-based adaptive evolution
Linjiang Zhu, Zhen Cai,Yanping Zhang and Yin Li
http://dx.doi.org/10.1002/biot.201300277
Research Article
Mini-scale cultivation method enables expeditious plasmid
production in Escherichia coli
Petra Grunzel, Maciej Pilarek, Dörte Steinbrück, Antje Neubauer,
Eva Brand, Michael U. Kumke, Peter Neubauer and Mirja Krause
http://dx.doi.org/10.1002/biot.201300177
Research Article
A magnetic nanobead-based bioassay provides sensitive detection of
single- and biplex bacterial DNA using a portable AC susceptometer
Mattias Strömberg,Teresa Zardán Gómez de la Torre, Mats
Nilsson, Peter Svedlindh and Maria Strømme
http://dx.doi.org/10.1002/biot.201300348
Research Article
Hydrostatic pressure and shear stress affect endothelin-1 and nitric
oxide release by endothelial cells in bioreactors
Federico Vozzi, Francesca Bianchi, Arti Ahluwalia and
Claudio Domenici
http://dx.doi.org/10.1002/biot.201300016
Technical report
A protease substrate profiling method that links site-specific
proteolysis with antibiotic resistance
Lisa Sandersjöö, George Kostallas, John Löfblom and
Patrik Samuelson
http://dx.doi.org/10.1002/biot.201300234
Rapid Communication
Albumin-based nanocomposite spheres for advanced drug delivery
systems
Heath E. Misak, Ramazan Asmatulu, Janani S. Gopu, Ka-Poh
Man, Nora M. Zacharias, Paul H. Wooley and Shang-You Yang
http://dx.doi.org/10.1002/biot.201300150
Biotechnology Journal – list of articles published in the January 2014 issue.
Our latest Biotech Methods & Advances special issue is edited by our Editors-in-Chief Prof. Alois Jungbauer
and Prof. Sang Yup Lee. As always, the special issue is a collection of the latest breakthroughs in biotechnology.
The cover is a graphical representation of some of the tools in biotechnology research. Image:
© Bank-Bank – Fotolia.com.
Systems & Synthetic Biology ·
Nanobiotech · Medicine
ISSN 1860-6768 · BJIOAM 9 (1) 1–170 (2014) · Vol.9 · January 2014
1/2014
Stem cells
Bioreactor
Plant biotech
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Methods & Advances