Review

Microfluidic Organ/Body-on-a-Chip Devices at the

Convergence of Biology and Microengineering
Ana Rubina Perestrelo 1, *, Ana C. P. Águas 2 , Alberto Rainer 3 and Giancarlo Forte 1,4, *
Received: 22 September 2015; Accepted: 4 December 2015; Published: 10 December 2015
Academic Editor: Sandeep Kumar Vashist
1 International Clinical Research Center (ICRC), Integrated Center of Cellular Therapy and Regenerative
Medicine (ICCT), St. Anne’s University Hospital, Brno 656 91, Czech Republic
2 Center for Biomedical Research, University of Algarve, Faro 8005-139, Portugal; [email protected]
3 Tissue Engineering Unit, Università Campus Bio-Medico di Roma, Rome 00128, Italy;
[email protected]
4 Department of Biomaterials Science, University of Turku, Turku 20014, Finland
* Correspondence: [email protected] (A.R.P.); [email protected] (G.F.);
Tel.: +420-543-185-526 (A.R.P. & G.F.); Fax: +420-543-182-100 (A.R.P. & G.F.)

Abstract: Recent advances in biomedical technologies are mostly related to the convergence of
biology with microengineering. For instance, microfluidic devices are now commonly found in
most research centers, clinics and hospitals, contributing to more accurate studies and therapies
as powerful tools for drug delivery, monitoring of specific analytes, and medical diagnostics. Most
remarkably, integration of cellularized constructs within microengineered platforms has enabled the
recapitulation of the physiological and pathological conditions of complex tissues and organs. The
so-called “organ-on-a-chip” technology, which represents a new avenue in the field of advanced
in vitro models, with the potential to revolutionize current approaches to drug screening and
toxicology studies. This review aims to highlight recent advances of microfluidic-based devices
towards a body-on-a-chip concept, exploring their technology and broad applications in the
biomedical field.

Keywords: microfluidics; BioMEMs; organ-on-a-chip; body-on-a-chip; tissue engineering

1. Introduction
Twenty years after the first definition of tissue engineering (TE) came out [1], tissue engineers
are now facing new challenges concerning the standardization of the production protocols, cost
reduction and up-scaling of these standardized procedures to the clinical setting. Most remarkably,
knowledge deriving from tissue engineering is finding increasing application in the development of
micro-engineered models of human tissues and organs, which are being investigated as potential
alternatives to animal models in elucidating the biological mechanisms underlying morphogenetic
and pathogenetic processes, as well as drug screening platforms [2–4]. In this scenario, tissue
engineering adds the third dimension (3D) to in vitro cell cultures, better mimicking the complexity
of native tissues and giving access to full-human models.
Major advances in this field are related to the integration of tissue engineering with
microelectronics, microfabrication and microfluidics. Electronic devices have been employed as
integrative systems for tissue engineering research. Biosensors, initially dedicated to the detection of
biomolecules such as proteins [5,6], peptides [7,8], enzymes [9,10] and DNA [11,12], are now proposed
in the tissue engineering field as tools to monitor cell behavior on a miniaturized scale, with high
sensitivity and resolution and low associated costs [13–15]. By detecting cellular analytes, electrical
activity, physical and chemical signals transmitted by the cells, biosensors can provide insights into

Sensors 2015, 15, 31142–31170; doi:10.3390/s151229848 www.mdpi.com/journal/sensors

Sensors 2015, 15, 31142–31170

cellular activities and responses in real time. Therefore microfluidic-based biosensors—also known as
lab-on-a-chip (LOC) and Biological/Biomedical Micro Electro Mechanical Systems (BioMEMS)—are
becoming more and more popular.
Microfluidic-based biosensors consist of devices in which the manipulation and analysis of fluids
occur within micrometer-sized channels [16,17]. Thanks to this miniaturization, the applications
of microfluidic devices are countless. To date, microfluidics has been successfully in monitoring
and controlling diagnostics [18], cell manipulation [19,20] and drug delivery [21]. Furthermore, the
most advanced microfluidic devices not only allow for the monitoring of signals but also actively
respond and adapt to them. Here, we highlight and summarize current cutting-edge research on
microfluidic devices, their application at a 3D level in tissue engineering and recent developments
towards body-on-a-chip concept.

2. Microfluidics—from Small Benchtop Biosensors to High-Throughput Systems

Although the concept of microfluidics is associated with a framework of complexity and
robustness, its roots date back to the 1950s, principally for what concerns inkjet printing technology.
As the name suggests, microfluidics is the science and technology associated to the control and
manipulation of liquids at a scale of few microliters. Due to the associated advantages of reduced
sample volume, scalability, laminar flow and hence highly predictable fluid dynamics, high resolution
and sensitivity, short time of analysis, and low cost, there are innumerous fields where microfluidics
can be useful and are actually applied.
In addition to faster medical diagnostics [22–25], microfluidics is being applied in drugs of abuse
testing [26–29], pollutant detection [30–34], combatting biowarfare [35–38] and also in laboratory
routines in a research context, as described below.
Microfluidics is bound to fit the needs of researchers mainly due to its high-throughput
capacity to scale up the number of assays in an automated manner and integration capacity in large
experimental pipelines, while reducing the costs. A good example of how this compromise is kept
is given by the microfluidic chromatographic column developed by Shapiro and collaborators to test
several separation conditions for biopharmaceuticals [39].
On the other hand, with the aim of reducing the experimental costs and reagent volumes
while keeping the high-throughput capacity of the system, Chen and Ismagilov have developed
an alternative to 96-well plates for drug screening, using microfluidic cartridges pre-loaded with
nanoliter plugs of reagents [40]. This technology could be applied to biological and chemical assays
with associated low cost and simplicity.
In addition, assays that require thermocycling could also be sped up by using microfluidics
technology, as reviewed by Zhang and Xing [41]. Short-term assays, low reagent consumption and
rapid heating/cooling rates are some of the advantages of miniaturized PCR devices, which are assets
in applications like the molecular diagnostics of diseases [42–44] and gene expression analysis [45–48].
Some of the drawbacks found in conventional research are related with sample manipulation,
destabilization of measured signals due to interventions to load a sample or to change a buffer,
coupled to the time-consuming experiments and mostly due to the lack of processes automation.
By integrating and automating standard laboratory routines, microfluidics technology allows to
overcome these limitations, saving time, resources and improving the quality and reproducibility
of the results. Indeed, instrument and protocol up-grade to integrate microfluidic platforms allows
incorporating several experimental setups into a simplest one with synchronized assay execution
and data record. Due to its great impact in research improvement and efficiency, this technology has
been adopted by several research groups, increasing the degree of complexity of the assays but also
yielding more reliable and reproducible results.
For instance, Mellors and collaborators developed a fully integrated microfluidic platform to
perform high efficiency capillary electrophoresis and electrospray ionization mass spectrometry
analysis, useful for proteomics applications [49]. On the other hand, Focke et al. described

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microfluidic cartridges for DNA purification and genotyping, by using standard laboratory
instruments as integrative systems [50].
New microfluidic platforms appear every day as a toolbox for the development of new solutions,
either to solve benchwork issues or to meet biomedical needs. The complexity and characteristics of
the systems obviously depend on the application-specific requirements, varying from very simple
devices to engineered complex platforms.
Paper-based microfluidics systems are the simplest technology for point-of-care diagnostics,
combining the well-known methods of lateral flow tests with paper microfluidic technology, where a
thin sheet of porous material is the substrate for the bioassays, taking advantage of the substrate high
internal surface area, capillary action and absorptive capacity [51]. Notably, the dramatic reduction
of costs brought in by paper-based microfluidics holds promise to bring point-of-care diagnostics to
developing countries [52].
Microfluidics finds an application in the standardization of cell culture protocols and in
the setup of reliable and sensitive bio-sensing assay protocols. Indeed, cells need optimal
physiological conditions (pH, temperature and CO2 ) to ensure their viability and activity, they must
be continuously and uniformly perfused with nutrients and oxygen and precautions are needed to
avoid biofouling effects [53] and side reactions due to non-specific adsorption of biomolecules [54].
Microscale fluid regulators as valves, pumps, mixers and other functional elements allow cell
perfusion with fresh media and assay reagents. Furthermore, automated liquid handling, electronic
control of switches and valves, multiplexing capability and appropriate detectors to monitor cellular
stimuli make a high-throughput screening format feasible. The wide range of different laboratory
activities, which already benefit from advanced systems in microfluidics-dependent cell assays, is
reviewed elsewhere [55,56].

3. Convergence between Microfluidics and Tissue Engineering: Bio-MEMS and Organ-on-a-Chip

BioMEMS are increasingly contributing to TE by providing accurate control of the cell
environment in settings suitable for cell screening and by enabling the engineering and studying
of minimally functional modules of complex tissues [57]. Although definitions are somewhat
overlapping, this last approach is also commonly defined as “organ-on-a-chip” (OoC).
In this chapter, we highlight the features of BioMEMs as in vitro models of cardiovascular,
respiratory, nervous, digestive, endocrine and integumentary systems and pathologies (Table 1,
Figure 1).

Table 1. Recent applications of BioMEMs.

Application Platform References

Cardiovascular System
Dual channel chip/angiogenesis model, microfluidic
Angiogenesis studies [58,59]
tri-culture platform
Pressure attenuator + Funnel chain/cell deformability
[60]
microfluidic device
Muscular thin films [61]
Biophysical studies
Microfluidics + optical microscopy [62]
Microfluidics + ultrasound imaging system [63]
High-speed video microscopy in microcapillaries [64]
Drug Microchannel microfluidic chip [65]
screening/development Laminar ventricular muscle-on-a-chip [66]
Microfluidic cardiac cell culture model, heart-on-a-chip,
artery-on-a-chip, microscale blood vessel module (µBVM) in
Organ/tissue a single microchannel device, microfluidic perfusion cell
[61,67–74]
structure/activity culture chip, microfluidic delivery system, microchannel
biochips as vaso-occlusive processes model, perfusion
microfluidic device, branched microfluidic channels

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Table 1. Cont.

Application Platform References

Respiratory system
Flow stretch chip [75]
Biological barriers
Compartmentalized microwells in a microfluidic device [76]
Cancer mechanisms Microfluidics + electric fields [77]
Cell culture Biomimetic microfluidic airway model [78]
3D gelatin-microbbuble scaffold produced by microfluidic
Cell differentiation [79]
device
Dynamic transwell microfluidic system + perfusion culture,
Cell migration [80,81]
microfluidic gradient generator
Drug delivery Microfludics + surface acoustic wave (SAW) nebulizer [82]
In vivo organ studies Microfludics + single oxygenator units [83]
Molecular mechanisms Microfluidics + concentration gradient generator [84]
Microfluidic system of converging multichannels +
Wound healing [85]
hydrodynamic flow focusing
Nervous System
Microchannels/microgrooves + compartmented
Axonal transport [86–89]
microfluidic culture
Microchannels/microgrooves + compartmented microfluidic
Cell culture [90,91]
co-cultures, shear-free microfluidic gradient generator
Microfluidics + electrophoresis [92]
Cell line characterization
Microfluidics + quantitative reverse transcriptase polymerase
[93]
chain reaction (qRT-PCR)
Microgrooves + neuronal compartment + myelination
Cell differentiation [94]
compartment microfluidic co-cultures
Microfluidic microgrooves + compartment to culture explants
Cell migration [95]
+ compartment with Matrigelr to receive migrating neurons
Two-compartment microfluidic culture system (neuronal
compartment + myelination compartment) microfluidic
Cellular/Molecular [94,96–98]
co-cultures, microfluidic axon-microglia platform, axon
mechanisms
injury micro-compression platform
Microfluidic devices or bioreactors + ultra-performance
[99]
liquid chromatography-ion mobility-mass (UPLC-IM-MS)
Drug delivery Microfluidic + perfusion device [100]
Microfluidic “Fish-Trap” array, gravity-induced flow +
[101,102]
Drug screening/development microfluidic chip
Microfluidics + trans-endothelial electrical resistance (TEER) [103]
Organ/tissue Microfluidic “Fish-Trap” array, two-compartment +
[90,101]
structure/activity microchannels microfluidic culture system
Screening / Diagnostic Microfluidic cell sorter [104]
Three compartment microfluidic device competition
Synaptic studies experiment, two cell culture chambers + funnel-shaped [105,106]
micro-channels microfluidic device
Axonal microfluidic chambers [107]
Toxicity studies
Microfluidics + 96-well plate [108]
Digestive + Excretory System
Biomimic hydrogel nephron [109]
Integrated Dynamic Cell Culture Microchip (IDCCM),
Microfluidic endothelial-like barrier, dam-wall and nozzle
[110–114]
microfluidic device, hemi-coaxial-flow channel microfluidic,
Cell culture dual perifusion platform
Microfluidic bioreactor [115–117]
Microfluidic droplet-based cell encapsulation [118]
Multiwell culture system [119,120]
Microfluidic-multilayer device (MMD) [121]

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Table 1. Cont.

Application Platform References

Microfluidic cell culture chamber/channels [122,123]
Cell differentiation
Microfluidics + qRT-PCR [124]
Microfluidic geometrically enhanced mixing chip,
Circulating tumor
Geometrically Enhanced Differential Immunocapture [125–127]
cells studies
(GEDI) device
Gut-on-a-chip, 3D villi scaffold + microfluidic
[128–131]
device, IDCCM
Drug Microfluidics + optical fiber [132]
screening/development Microfluidic cell culture array [133]
Microfluidic droplet-based cell encapsulation [118]
Three-dimensional microfluidic microanalytical micro-organ
[134,135]
device (3MD)
Food analysis Microfluidics + Fluorescence imaging [136]
IDCCM, two-plate bioreactor, metabolomics-on-a-chip,
[110,117,131,
microfluidic delivery device, two-color detection microfluidic
137–140]
system, multimodal islet hypoxia device
Metabolism studies Microfluidic bioreactor [141]
Microscale cell culture analogue (µCCA) [142]
Microfluidics-optical sensor [143]
Multiwell culture system [119]
Integrated Insert in a Dynamic Microfluidic Platform
Organ-organ interaction (IIDMP), on-chip small intestine-liver coupled microfluidic [144,145]
network
Microfluidics + surface plasmon resonance [146]
Screening/Diagnostic Microfluidics + optoelectronic sensor [147]
Microfluidics + optomechanical metric [148]
Therapeutic systems Wearable ultrafiltration units for dialysis [149,150]
Metabolomics-on-a-chip, Gut-on-a-chip, IDDCM bioreactor,
[137,151–154]
pharmacokinetic microfluidic perfusion system
Kidney and kidney/liver microfluidic biochips [155–157]
Microfluidics + optical fiber [132]
Toxicity studies µCCA [142,158]
Microfluidic bioreactor [159]
Microfluidic human kidney proximal tubule-on-a-chip device [160]
MMD [121]
Multiwell culture system [119]
Endocrine System
Microfluidic co-culture model, chemokine gradient + 3D
Cancer mechanisms [161,162]
culture device
Motile spermatozoa sorter + microfluidic chip, microfluidic
Fertilization [163,164]
device mimicking female reproductive tract
Metabolism studies Microfluidics + resonant waveguide grating (RWG) sensor [165]
Monitoring Microfluidics + electrochemical sensor [166]
Blood plasma separation microfluidic chip [18]
Microfluidics + optical sensor [167]
Screening and Microfluidics + liquid chromatography-mass spectrometry [168,169]
diagnostic Microfluidics + potentiostat [170]
Microfluidics + electrochemical sensor [171]
Digital microfluidics [172]
Integumentary System
Biological barriers Stable gel/liquid interface microfluidic chip [173]
Cell differentiation Pillar array microfluidic device based on cell surface markers [174]
Cell migration 3D matrices microfluidic device [175]
Screening and Microfluidics + conductometric sensor [176]
diagnostic Microfluidics + potentiometric sensor [177]
Microfluidic wound-healing model + wound
Skin repair [178,179]
dressing screening

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In general, such devices are obtained by soft-lithographic processes, with polydimethylsiloxane

(PDMS) and glass representing common materials for the fabrication of microfluidic channels,
which makes such devices compatible with live-cell microscopy and high throughput
screening methodologies.
Devices may also endow porous membranes to compartmentalize different cell populations and
biomimetic coatings with extracellular matrix (ECM) components such as fibronectin, collagen, or
Matrigelr to improve cell attachment. A comprehensive review of biomaterial-related issues for the
fabrication of BioMEMs has been provided by Berthier et al. [180].

3.1. Cardiovascular System

More than in the direct treatment of cardiovascular pathologies, microfluidics strategies and/or
devices are being applied in in vitro models, diagnostics, clinical studies and drug screening with
the aim of reducing the intervention time and to set up more efficient therapies. Thanks to
their conduit-like design, and to their precise control over flow conditions, including shear stress
and pulsatility, microfluidic devices are particularly likely to be used as reductionist models of
cardiovascular biology (e.g., to mimic blood flow and predict injuries to blood vessels), than to
study heart-related issues. Nevertheless, modern biomedical engineering is advanced enough as to
reproduce cardiovascular system complexity. Microfluidic cardiac cell cultures are physiologically
relevant in vitro models that recreate mechanical loading conditions seen in both normal and
pathological conditions and allow hemodynamic stimulation of cardiomyocytes by directly coupling
cell structure and function with fluid-induced loading [61,67]. In this context, an example of
“heart-on-a-chip” was given by using poly(N-isopropylacrylamide) (PIPAAm) and PDMS to engineer
an anisotropic rat ventricular tissue and to measure contractility, action potential propagation,
epinephrine dose-response and cytoskeletal architecture in a mid- to high-throughput system
allowing real time data collection [61].
Exploiting a similar PIPAAm/PDMS-based system, the same research group provided evidence
that it is indeed possible to reproduce on chip the negative remodeling of the failing myocardium by
applying cyclic mechanical stretch to mimic pathological mechanical overload [66].
Furthermore, if combined with cells or biopsies harvested from patients, these models could
be used as tools for drug screening in individualized medicine. The major concern with the
setup of microfluidics systems for cardiovascular testing is in the peculiar growth attitude of
the cardiomyocytes, requiring special conditions to adhere and survive while preserving their
unique contractile phenotype. Thus, the discussion is still open to find the most appropriate and
representative source of contractile cells.
As soon as the vascular component of the cardiovascular system is taken into account,
reproducing the complexity of the system itself becomes more and more challenging. Several research
groups are interested in the development of microfluidic devices in which angiogenesis [58,59],
artery structure [68] and network [69], vascular endothelial function [70] growth and remodeling [71]
can be studied. More directed to vascular pathologies, other groups are focused in highlighting
vaso-occlusive processes [72] (Figure 1A) and thrombosis [73], evaluating hypertensive micro
vessels [74] and antihypertensive drug effects [65], or studying long-term vascular contractility [71].
Numerous blood pathologies are caused by the decrease of red blood cell deformability
impeding the transit of these cells through the microvasculature, where they play a central role in
the oxygenation of tissues. Therefore, a common indicator of hemorheological dysfunction is the
measure of red blood cell deformability or dynamic analysis of blood flow. Biophysical properties
including red blood cells aggregation, deformability, viscosity, velocity profile and pressure of blood
flows have been measured in systems engineered by Yeom [62,63], Guo [60], Tomaiuolo [64] and
Zheng [181,182] and were useful for understanding the effects of hemorheological features on the
hemodynamic characteristics in capillary blood vessels.

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3.2. Respiratory System

Most frequent respiratory diseases act by affecting the airways, the structure of the lung tissue,
blood circulation in the lungs, or involve a combination of these three. Given the precise control
over fluidic parameters, and the successful modeling of tissue interfaces, microfluidic platforms are
finding increasing application in the study of respiratory system pathophysiology.
Some of the first studies have reported biomimetic microsystems reproducing the
alveolar-capillary interface of the human lung as an alternative to animal and clinical studies, for
drug screening and toxicology applications [75,183,184] (Figure 1B). Since then, several authors
have developed biomimetic models, BioMEMs or microfluidic-based devices with the purpose of
highlighting and modeling important issues in lung development, differentiation, homeostasis and
disease [185]. Recently, two approaches used microfluidic devices to study the differentiation of
lung stem/progenitor cells in the view of future lung tissue engineering applications [79,186]. In
the first approach, alveoli-like structures were obtained after seeding isolated mouse pulmonary
stem/progenitor cells in a compatible gelatin/microbubble-scaffold using a 2-channel fluid jacket
microfluidic device [79]. The second strategy consisted in the development of microfluidic magnetic
activated cell sorting system in the isolation of mouse lung multipotent stem cells for further
characterization [186]. Different research groups are focused on the development of models that
mimic lung barrier and, in combination with cells from patients, are proposed as drug-screening
platforms to select candidate drugs to treat pulmonary pathologies [76,78].
As far as the onset of lung diseases is concerned, researchers are focused in producing
biomimetic microsystems so that the molecular processes underlying pathologies such as
malignant transformation of bronchial epithelial cells due to tobacco [84], protein-induced lung
inflammation [80], chronic obstructive pulmonary disease [81] and idiopathic pulmonary fibrosis [85]
can be highlighted.
Applications of microfluidics also regard the development of implantable respiratory assist
devices with a potential for clinical application. As an example, in the last few years, Kniazeva and
Hoganson described a small-scale microfluidic artificial lung and an implantable ambulatory lung
assist device based on stacked microchannel networks, ultrathin gas exchange membranes, and with
the potential to be used in the clinics [187–190].
While microfluidic artificial lung is still under development, several miniaturized devices are
now closer to being translated to the clinical application. Cortez et al. developed a portable
acoustomicrofluidic device capable of nebulizing drugs into a fine aerosol for deep lung deposition
via inhalation with negligible drug degradation, as successfully demonstrated in the case of
epidermal growth factor receptor (EGFR) monoclonal antibodies [82]. Also, Rochow engineered
a miniaturized oxygenator device, composed of stacked single microfluidic units and perfused
like an artificial placenta via the umbilical vessels, that might support newborns with respiratory
insufficiency [83].

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Figure 1. Illustration of the diverse microfluidic devices used to study biological processes
Figure 1. Illustration of the diverse microfluidic devices used to study biological processes
occurring in vascular, respiratory, nervous, digestive and excretory systems. A. Biochip with
occurring in vascular,
subdividing respiratory,
interconnecting nervous,(array
microchannels digestive andthat
of pillars) excretory
decreasesystems.
in size toA. Biochip
mimic cell with
flow and adhesion in microvasculature to study of vaso-occlusive processes. B. Human breathing
subdividing interconnecting microchannels (array of pillars) that decrease in size to mimic
lung-on-a-chip microdevice, a biomimetic microsystem that reconstitutes the alveolar-capillary
cell flow and ofadhesion
interface the lungs.inThe
microvasculature to study ofchambers
device uses compartmentalized vaso-occlusive
to form anprocesses. B. Human
alveolar-capillary
barrierlung-on-a-chip
breathing on a porous membrane and produces
microdevice, cyclic stretching
a biomimetic of such membrane
microsystem by vacuum
that reconstitutes the
actuation. C. Two-compartment microfluidic culture system bridged by microchannels. It allows the
alveolar-capillary interface of the lungs. The device uses compartmentalized chambers to
visualization of cell interactions in co-culture, namely as a model for synaptic connectivity between
form an mixedalveolar-capillary
hippocampal co-culturesbarrier on a microgrooves
in which porous membraneallow bothand axons produces cyclic
and dendrites stretching
to enter and of
form synapses. D. Vertical cross-section representing the on-chip generation of intestinal villi obtained
such membrane by vacuum actuation. C. Two-compartment microfluidic culture system
by villus morphogenesis of Caco-2 cells. The up-scale of this system leads to the production of
bridged by microchannels.
gut-on-a-chip It allows
platforms to study the visualization
pharmacokinetics of cell interactions
and diffusion processes. E. Artificial liverin co-culture,
sinusoid
namely withasa microfluidic
a model for synaptic connectivity
endothelial-like between
barrier for primary hepatocyte mixed
culturehippocampal
to study diffusiveco-cultures
nutrient in
transport in liver-mediated metabolism. This unit consists of a cord of hepatocytes fed by diffusion
whichofmicrogrooves allow both axons and dendrites to enter and form synapses. D. Vertical
nutrients across the narrow microfluidic channels from a convective transport vessel. F. Kidney
cross-section representing the
proximal tubule-on-a-chip. on-chip generation
The microfluidic device consists ofof intestinal villi separated
an apical channel obtainedfrom bya villus
bottom reservoir by a porous membrane upon which primary human proximal
morphogenesis of Caco-2 cells. The up-scale of this system leads to the production of gut- tubule epithelial cells
are cultured in the presence of apical fluid shear stress. This design mimics the dynamically active
on-a-chip platforms
mechanical to study pharmacokinetics
microenvironment and diffusion
of the living kidney proximal tubule and processes. E. Artificial
allows the study of active liver
sinusoidand with
passivea epithelial
microfluidic endothelial-like barrier for primary hepatocyte culture to study
transport.

diffusive nutrient transport in liver-mediated metabolism. This unit consists of a cord of

3.3. Nervous System
hepatocytes fed by diffusion of nutrients across the narrow microfluidic channels from a
Nervous system pathologies have their origin in aging, genetic alterations, brain trauma and
convective
spinal cord transport vessel.
injuries, among F. Kidney
others. Given theproximal
intrinsic tubule-on-a-chip.
complexity of the nervous The microfluidic
system, one of device
the
consists of an apical
most described channel
applications of separated
microfluidicfrom a bottom
technology reservoir by
is represented by ainporous membrane
vitro models mimicking upon
the nervous
which primary tissue-vasculature
human proximal interaction. Microfluidic
tubule epithelial cellsplatforms
are cultured have inbeen
the described
presence as of ideal
apical
in vitro cerebrovascular models not only due to their automatized features, miniaturized scale and
fluid shear stress. This design mimics the dynamically active mechanical microenvironment
low cost, but also because of their ability to mimic physiological dynamics, physical properties
ofand
thebiological
living kidney proximal tubule
microenvironment and allows
complexity [191]. the study of
Generally, suchactive andcan
devices passive epithelial
be applied to
model
transport. and study the progression of neurodegenerative diseases and screen drug candidates towards
individualized medicine solutions.

3.3. Nervous System

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Nervous system pathologies have their origin in aging, genetic alterations, brain trauma and spinal cord
injuries, among others. Given the intrinsic complexity of the nervous system, one of the most described
Sensors 2015, 15, 31142–31170

In particular, the impairment of blood-brain barrier (BBB) is considered to be among the

main causes of pathogenesis and/or progression of several neurological disorders such as epilepsy,
multiple sclerosis, Parkinson and Alzheimer’s disease. Therefore, a better understanding of the
physiology, microenvironment, cell-cell interactions at the BBB level can provide important clues
on brain disorders or help designing and testing efficient drug candidates. In 2012 Booth upgraded
the static (transwell) in vitro model of BBB to a dynamic one, and then used it to analyze neuroactive
drugs [103,192], establishing a versatile model for prediction of BBB clearance of pharmaceuticals.
In normal physiologic conditions, microfluidics-based in vitro models can contribute to a better
understanding of mechanisms behind the formation and function of neuronal networks. Thus, these
models allow for the reproduction of synaptic competition [105], cell line authentication [92,93],
study of neuronal migration in embryonic brain explants [95], axonal guidance during brain
development [93] (Figure 1C) and myelination [94]. The use of brain explants within microfluidic
devices also allows for the exposure to multiple compounds at once or in sequence, thus
improving the existent models towards an individual medicine approach as a guided therapeutic
decision-making [100,104,108]. Of great interest is the possibility to exploit microfluidics for
high-throughput mapping of brain-wide activity in awake and drug-responsive vertebrates (e.g.
zebrafish) [101].
To visualize the fundamental physiological changes occurring during the onset of
neurodegenerative diseases, microfluidic systems were developed to model synaptic connectivity
between mixed hippocampal co-cultures [90] (Figure 1C), to reconstruct neuronal network and
test β-amyloid toxicity [102,106] as well as to follow the activation of developmental brain
disorders [99]. At the axonal level, structural and functional deficits are predictive of an early
occurrence of neurodegenerative diseases. Therefore several platforms have been designed to
highlight mechanisms of axonal function impairment [97], axon-polarization [91], axon toxicity [107],
deformation [98], and to trace axonal transport at single vesicle level [86–89].

3.4. Digestive and Excretory Systems

A variety of diseases negatively affecting digestive system lead to gastrointestinal organ damage
and function deterioration. Stomach and esophagus cancer, short bowel syndrome, fecal incontinence
and trauma are among the pathologies affecting gastrointestinal function and urging for a treatment.
In the early diagnosis context, Zilberman and Sonkusale stood out with a strategy based on
optoelectronic sensors for early gastric cancer detection in saliva, thus proposing an alternative
non-invasive method to endoscopy, biopsy and histopathological evaluation [147].
Since conventional 2D culture systems lack reproducibility of chemical complexity and
biofunctionality of the living tissues, microfluidics arose as an alternative platform to develop
strategies to settle gastrointestinal tissue regeneration and study organ physiological functionality.
Due to the structure and dynamic features of BioMEMs, there is a great interest in the use of these
systems to more accurately study the intestinal absorption of drugs and their toxicity. For instance,
Kimura and colleagues developed an integrated microfluidic system endowing on-chip pumping and
optical fiber detection systems. Performance of the device was examined through long-term culture
and monitoring of polarized transport activity of Caco-2 cells [132]. Mahler et al. [158], as well as
McAuliffe and collaborators [142], have also contributed to the design of drug transport models using
microfluidic devices.
Microfluidic complex systems to create in vitro models of the intestine are valuable tools to study
gut function under normal or diseased conditions and also to perform drug screening and toxicity
assays. In this regard Kim and co-workers developed a microengineered “human gut-on-a-chip”,
a system composed by two microfluidic channels with a flexible porous membrane coated with
extracellular matrix, lined by gut epithelial cells (Caco-2), making it possible to recreate the gut
structure with its mechanical, absorptive, transport and pathophysiological properties [151]. One
year later, Kim and Ingber demonstrated that applying specific physiological mechanical cues

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to the gut-on-a-chip, it was possible to induce Caco-2 cells to spontaneously undergo intestinal
villi morphogenesis [128] (Figure 1D). This model was recently upscaled by the development
of a platform that can be adaptable to produce several functional units of other organs [152].
Further improvements in the recapitulation of the “intestinal epithelium-on-a-chip” consist in
the fabrication of 3D-shaped microporous polymeric membranes mimicking the geometry of the
intestinal villi [129] or by the so called “intestinal epithelium-on-a-chip” being reproduced by using
a novel hydrogel microfabrication technique and showing a superior structural maturation [130].
Their microfluidic device was further used to study the kinetics of diffusion processes in the 3D villi
scaffold. A much higher degree of complexity was reached by Ramadan and collaborators with the
microfluidic platform called NutriChip. With the aim to analyze the passage of nutrients through the
gastrointestinal tract (GIT), they developed a miniaturized GIT including the epithelial and immune
cell components, in which the response of immune cells to pro and anti-inflammatory stimuli was
monitored [130].
The liver also plays an important role in the digestion processes, being responsible for
the filtration of nutrients and digestion products. Furthermore, liver represents a fundamental
key in the metabolism of xenobiotics and thus a number of publications came out in the last
years, describing strategies to recreate liver-specific functions through microengineered models,
with the purpose of studying drug metabolism and ultimately improving drug development
processes [118–120,134,135]. Moreover, numerous publications described the use of microfluidics
for the development of physiologically-relevant hepatocyte cell culture [110,111,115,117] (Figure 1E),
differentiation [122] and co-culture systems [117,132,144,193] as well as the design of platforms
for diagnostic applications [146]. Additionally, the development of microfluidic-based devices to
investigate liver drug metabolism and toxicity [131,133,137,153,154,159,194] are to be considered
fundamental tools to address liver pathologies, better understand molecular toxicity mechanisms
and simulate drug-drug and organ-organ interactions. A comprehensive review about this topic is
given by van Midwoud and colleagues [195].
Pancreas also plays an important role in the digestion process, as it is responsible for
producing enzymes and hormones to be secreted into the small intestine. Deficient production of
digestive enzymes and hormones, as well pancreas blockage by tumors and gallstones, leads to
subsequent malfunction of the entire digestive system and further compliances. Trying to solve
these life-threatening conditions, researchers have made use of microfluidics to study and diagnose
pancreatic cancer [125–127,148], culture pancreatic islets [112–114,138], monitor stimulus-secretion
factors [139,140,143] and promote tissue-specific cell differentiation [124].
The perfect example of how microfluidics can be successfully applied to treat pancreatic
dysfunctions comes from the “bionic pancreas” developed for type 1 diabetes, that uses continuous
glucose monitoring along with subcutaneous delivery of both rapid-acting insulin and glucagon to
lower/increase blood glucose levels [196].
Due to the important functions on processing digestion products, water balance and blood
pressure regulation, kidneys are of fundamental importance for whole-body homeostasis. Therefore,
there is a considerable interest to develop strategies to adequately treat the most problematic
conditions affecting kidneys. Among them, chronic kidney disease often results in end-stage renal
failure, requiring renal replacement therapy and eventually transplantation, causing a massive
burden on the healthcare systems. Microfluidic systems, as the one developed by Leonard and
collaborators, appear as innovative tools to improve the outcome of classical approaches [150,151].
For instance, a membraneless dialysis strategy was developed, opening possibilities to create
wearable blood processing devices [150,151]. Other microfluidic systems enable the culture of kidney
cells in tubular structures, mimicking the organ structure and function [109,121,123,160] (Figure 1F).
The potential of application of microfluidic also includes disease modeling and metabolism studies,
giving insights about kidney cell toxicity and renal clearance [141,155,156,160]. Renal excretion and
metabolism are the actual subjects of preclinical safety studies, with the goal of investigating drug

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pharmacokinetics in in vivo-like pathophysiological conditions. Therefore, microfluidic devices can

be useful to co-culture different cell types [157] with particular impact on the recreation of multi-organ
systems to study systemic interaction where kidneys and also liver can be incorporated. A wide
perspective on multi-organs-on-a-chip is described in Section 4.

3.5. Other Promising Applications for Microfluidics Technology

Although being still in their very early developmental stage, some microfluidic platforms appear
as innovative systems for significant endocrine studies. Concerning the adrenal glands, microfluidics
is being applied to detect and study corticosteroids [167,168,170] and catecholamines [165,166,171].
In the fertility context, Huang and collaborators used microfluidics to isolate, analyze and quantify
spermatozoids [163]. By a similar approach, Tung and collaborators demonstrated that the
biophysical environment of female reproductive tract critically guide sperm migration without
aiding the migration of pathogens [164]. Kim, Broccardo and co-workers used microfluidics to
quantify steroid hormone levels in tissue [169] and in human serum [168], what can be relevant
in fertility and osteoporosis studies. Microfluidic systems have been also employed with the
aim to diagnose thyroid diseases, as described by Shamsi and co-workers as well as by Madadi
and colleagues [18,172]. Further studies employing microfluidics platforms were performed in
hormonally-responsive cancers. Lang and colleagues explored breast cancer microenvironment
activity using protein levels as a sensor to predict how cell signaling is related with the growth of
cancer cells [161]. On the other hand, Kim and co-workers examined how chemoinvasion processes
are affected by chemical gradients, studying tumor cell migration behavior to understand the first
steps of cancer metastasis [162].
Microfluidics also appears as an innovative application in the wearable sensors for continuous
physiological signals monitoring. Sweat, as a non-invasive biofluid, is the subject of intense
investigations in this context. For instance, Rose and collaborators, as well as Liu and colleagues,
developed sensor patches for sweat electrolytes monitoring and aiming at hydration control [176,177].
In turn, Xu and co-workers described experimental and theoretical approaches for soft microfluidics
assemblies in sensors, circuits and radios for the skin [197]. Furthermore, the group of Sonner
has recently reviewed microfluidics models for eccrine sweat generation and flow, as a guide for
sweat-based diagnostics development [198]. Other recent approaches to investigate the function
and deficits of integumentary system comprise the microfluidics platform developed to study the
accumulation of molecules at the basal lamina interfaces and achieve efficient drugs and carriers’
distribution through biological barriers [173]. Microfluidics applications to model skin diseases
and for skin tissue regeneration are still in an early stage. However, some works in wound
healing [174,178,179] and cell migration [175] showed that this technology may have potential to
treat skin injuries.

4. Body-on-a-Chip: A Future Perspective

According to a recent analysis by Scannell et al., the past 60 years have seen huge advances
in many of the scientific, technological and managerial factors that should tend to raise the
efficiency of commercial drug R&D [199]. Yet the number of new drugs approved per billion
US dollars spent on R&D has halved roughly every nine years since 1950, falling around 80-fold
in inflation-adjusted terms. Improving the effectiveness of preclinical predictions of human drug
responses is critical to reducing costly failures in clinical trials. As evidenced in the previous
section, recent advances in tissue engineering, microfabrication and microfluidics have enabled the
development of microengineered models of the functional units of human organs. This approach is
believed to provide the basis for preclinical assays with greater predictive power [3]. This concept can
be further extended, recapitulating the function of several organs on a single microfluidic platform,
with the final goal to mimic the whole body physiology. Therefore, the Body-on-a-Chip (BoC) concept
is gaining relevance as a suitable device to study and predict cell-drug and cell-cell response [200].

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BoC devices consist of microfluidic chips into which several modules can be installed holding
different cell types or engineered human organs [201]. Samples are interconnected in a hierarchic
and physiologically relevant fashion, thus allowing the functional modeling and monitoring of the
circulatory, endocrine, digestive, immune, lymphatic, nervous, respiratory and urinary systems, as
an advanced human in vitro model (Figure 2).
Since BoC models mimic physiological context and key aspects of human metabolism, they
allow for:

‚ high accuracy prediction and comprehensive analyses of novel therapeutic candidates during
preclinical stages, by a closer estimation of efficacy and dose response;
‚ reduction and likely replacement of animals in preclinical drug development, thereby reducing
costs and time to market;
‚ creation of a drug development tool that helps modern medicine rapidly respond to fast-moving
pandemics or chemical warfare/bioterrorism attacks;
‚ study cell signaling by monitoring the metabolites that are consumed, produced, and exchanged
between all tissues at physiologically relevant concentrations in real time;
study
Sensors
‚ 2015,embryology
15 and its signaling pathways by following intercellular signals and/or 15
bioelectrical messages;
‚ conduct
conductexperiments
experimentsthat
thatcannot
cannotbebeperformed
performedinincell
cellculture,
culture,e.g.,
e.g. study
study of
of tissue-tissue
tissue-tissue
interactions that occur as a result of metabolite travelling from one tissue to other distant tissue,
interactions that occur as a result of metabolite travelling from one tissue to other distant tissue,
and through dynamic forces that resemble blood circulation;
and through dynamic forces that resemble blood circulation;
‚ efficient and reliable cell–cell and cell-drug/biomaterial interaction studies, narrowing the gap
between
efficient and reliable cell–cell and cell-drug/biomaterial interaction studies, narrowing the gap
in vivo and in vitro conditions.
between in vivo and in vitro conditions.

Figure 2. Schematic representation of a BoC approach in which cell-autonomous and non-autonomous

Figure 2. Schematic representation of a BoC approach in which cell-autonomous and non-
studies can be performed using a single chip.
autonomous studies can be performed using a single chip.

Microfluidics can bring more benefits if complemented with sensitive analytical methods (namely
mass spectroscopy and sensors), enabling the31153 metabolic profiling and comprehensive molecular
characterization of the chip-based cell systems. Furthermore, as the chip channels are usually
transparent, it is also possible to monitor cell response and perform cell-tracking through time lapse live-
Sensors 2015, 15, 31142–31170

Microfluidics can bring more benefits if complemented with sensitive analytical methods
(namely mass spectroscopy and sensors), enabling the metabolic profiling and comprehensive
molecular characterization of the chip-based cell systems. Furthermore, as the chip channels are
usually transparent, it is also possible to monitor cell response and perform cell-tracking through
time lapse live-cell imaging.
It is important to recognize that there are two complementary approaches for BoC development.
Bottom-up approaches start from a detailed specification of each organ, and then proceed with
the design of coupled systems (e.g., heart–lung and intestine–liver), adding organs to create more
complex models. Top–down approaches, on the contrary, consider the abstract, system-level
architecture of an organism and then break the system down into the functionality of compositional
organ systems.
Moreover, it is also possible to explore the process of inflammation response by adding cytokines
or living immune cells to the system [202,203]. Also, BoC devices with biopsy samples or cells from
individual patients can be very helpful in the development of individualized medicine to predict how
the patient might react to a certain pharmacological treatment, prior to administration, thus reducing
risks [204].

4.1. Proposed Applications of BoCs

As listed in Table 2, several BoCs are currently being developed. For instance, BoC simulation
with gastrointestinal tract and liver tissues was prepared by co-culturing Caco-2 enterocytes,
TH29-MTX mucin-producing cell line and HepG2/C3A hepatocytes in a microfluidic device. The
results suggested that ingested carboxylated polystyrene nanoparticles have the potential to cause
liver injury, thus showing that BoC devices are highly relevant in vitro multicellular models for
evaluating nanoparticle interactions with human tissues [200].
Vunjak-Novakovic and her team developed the HeLiVa platform, an integrated
heart-liver-vascular system derived from a single line of human pluripotent stem cells and
enabling the functional representation of human physiology in combination with real-time biological
readouts and compatibility with high-throughput analysis [205]. In the same pharmacological
context, the first pass intestinal and liver metabolism of paracetamol in a microfluidic platform
coupled with mathematical modeling as a means to evaluate absorption, distribution, metabolism,
and excretion (ADME) processes in humans was described by Prot and collaborators [206]. The
overall approach provided a first step in an integrated strategy combining in silico and in vitro
methods based on microfluidics for evaluating drug ADME processes [206]. Approximately one year
later, a four-organ-chip for interconnected long-term co-culture of human intestine, liver, skin and
kidney equivalents was introduced [207]. The system guarantees near to physiological fluid-to-tissue
ratios and the establishment of reproducible homeostasis among the co-cultures, sustainable over at
least 28 days. This system thus qualifies as a powerful tool to perform in vitro microfluidic ADME
profiling and repeated dose systemic toxicity testing of drug candidates [208]. Pursuing to design
a system with a higher degree of complexity, a 96-well format-based microfluidics platform was
prepared to interconnect various multicellular 3D spheroids, which enables parallelized culturing
and testing of spherical microtissues of different cell types in a standard incubator [209]. This
kind of device allows for the study of tissue-tissue interactions in the presence of pharmacologic
components [210,211]. According to the manufacturer, Swiss startup InSpheror , the commercial
multi-tissue device could be ready in three years.
Exploiting the possibility to connect different chips to increase the physiological relevance of the
in vitro system, efforts are being made in recreating whole human body by bridging the fluidics of
multiple chips, so that the physiological pharmacokinetics of the drugs of interest can be studied in a
very complex, representative in vitro system.

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Table 2. Available BoCs and their applications.

Organs /
Device / Platform Name Application References
Interactions
Brain, Heart, Lung, ADME profiling and
Physiologically-based
Skin, Adipose, quantification of the amount of
pharmacokinetic [212,213]
Muscle, Liver, Bone drugs in different parts of
(PBPK) model
Marrow, Kidney the body
Evaluating nanoparticle toxicity
Gastrointestinal µCCA [200]
and interactions with tissues
Tract and Liver
Investigate paracetamol intestinal
Gut—parallel tube model [206]
and liver first pass metabolism
Heart, Liver,
HeLiVa Drug testing in human health [205]
Vascular System
and disease
Intestine, Liver,
Four-Organ-Chip ADME profiling and [207]
Skin and Kidney
toxicity testing
Liver, Colorectal 96-well format-based
Testing drug effects at different [209–211]
Tissues microfluidic platform
concentrations in several tissues
Liver, Heart, Lung
ATHENA (“Homo Minutus”) Screening new drugs for potency [214]
and Kidney
and potential side-effects
Pharmacokinetic-pharmacodynamic Testing drug toxicity and
Liver, Tumor and
(PK-PD) model combined with improve insights into the drug’s [212]
Marrow
a µCCA mechanism of action
Lung, Gut PDMS-based organs-on-chip Prediction of clinical responses in [152,215]
humans

In a first approach, this pharmacokinetics–pharmacodynamics platform was tested with three

cell lines representing the liver, tumor and bone marrow [212], but can also be extrapolated for more
organs to predict mammalian response to drug and chemical exposure [213].
With a similar aim, the group of Donald E. Ingber is designing organs-on-chip which replicate
key functional units of living organs to reconstitute integrated human organ-level pathophysiology
in vitro [152,215]. The final purpose will be to combine as many organs as possible to closely mimic
a real human body. The development of the ATHENA (Advanced Tissue-engineered Human Ectypal
Network Analyzer) platform, also known as “Homo Minutus”, in which four interconnected human
organ constructs (liver, heart, lung and kidney) are interconnected in a highly miniaturized platform
follows the same principle [214]. This “Benchtop Human” is a big promise as it has the ability to
simulate the spatial and functional complexity of human organs, leading to a more accurate way of
screening new drugs for potency and potential side effects than current methods [216].

4.2. BoCs and Cancer

The interest in disclosing the signals peculiar of the cancer microenvironment and influencing
tumor cell growth, malignancy [217–221] and transvascular migration [222,223], is growing steadily
together with the search for new cancer prevention and diagnostics tools [224]. Scientists are aware
that tumor cell migration and intravasation into capillaries is an early and key event in cancer
metastasis. Therefore, several platforms have been developed with the aim to efficiently detect and
harvest circulating tumor cells (CTCs) and clusters, and for chemosensitivity or chemoresistance
assays. In fact, several works report the development of microfluidic devices for the isolation
of CTCs from lung [221,224–227], pancreatic [125,126], breast [217,226,228], ovarian [217,229],
prostate [217,229,230], colorectal [231], gastric [228], hepatic [232] and skin (melanoma) [229] cancer.
Concerning lung cancer, it is possible to characterize the early stages of progression while
predicting the occurrence of metastasis using CTCs from patients [233]. Also, to provide an
inexpensive and effective tool for CTC detection and evaluation of cancer status, Huang and

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collaborators developed a microfluidic size-based sorting platform, with the advantage of capturing
tumor cells without taking into account the expression of specific cell surface markers [234].
With a different aim, Ying and collaborators fabricated a 3D microfluidic chip generating
a concentration gradient of hepatocyte growth factor (HGF) to investigate its impact on Met/
PI3K/AKT activation, glucose regulatory protein expression and paclitaxel-induced A549 cell
apoptosis [235], as to mimic the in vivo secretion of the growth factor by cancer-associated fibroblasts.
Also, in order to modulate chemotaxis and electrotaxis of lung cancer cells, Kao employed
direct-current electric fields in a microfluidic cell culture device obtaining both stable electric field
and concentration gradients [77]. Recently, trying to be closer to a personalized medicine approach,
Ruppen and collaborators demonstrated the possibility to reproduce, at least partly, the barrier
induced by the tumor microenvironment to protect the tumor from drug exposure by testing the
chemosensitivity of patient lung cancer cell spheroids in a perfused microfluidic platform [236].
Phenomena such as tumor extravasation and metastatic site specificity have also been investigated
using 3D microfluidic models [237–239].
Several systems allow for the capture of CTCs and clusters from blood samples for further
detailed analysis of biomarkers by flow cytometry technology and multi-imaging [205,221–223].
Furthermore, the capture of these cells/clusters is suited for the identification of patients with
metastatic cancer and RNA sequencing of cancer cells can elucidate about the presence of cell
mutations [226] and identify the tumor origin.

4.3. Limitations of BoCs

As thoroughly analyzed by Wikswo et al. [240], abstracting the complexity of biology to obtain
a meaningful model for studying the properties of the entire system poses significant challenges.
Determining the proper size of each organ and its perfusion conditions, vascularizing organ units
with proper surface-to-volume ratio, and integrating the system with models describing the states of
health and disease, are but a few problems that need to be solved for successful implementation of
BoC approach [213,214,240].
From a more operational perspective, due to their (micro) scale, BoCs show several limitations,
mainly related to the growth of cells in such tiny channels, the formation of air bubbles in the cell
culture channels and the hurdles of long-term experiments [152]. Furthermore, the use of 3D organs
within the chips is subjected to a high batch-to-batch variability. One other drawback is in BoCs being
usually complex systems that require a special skilled operator, so—as to favor their spreading and
use on a daily basis—they should be re-designed to be simple, flexible and user-friendly, in order
to be employed as benchtop analyzers. In addition, the effects that polymers and fluids utilized in
BoCs exert on cell behavior and in the adsorption of metabolites are still poorly understood. That
is why one of the challenges scientists will have to address while trying to set up physiological
in vitro models of diseases by BoCs technology is to establish suitable universal cell culture conditions
(a universal ‘blood surrogate’) enabling the preservation of cellular phenotype and function, and
providing effective humoral communication between the different cell and tissue types [240].
Despite all the barriers to reach commercialization, there are several devices already approved
and/or under approval by FDA. For instance, CellSearchr is considered the first FDA-approved
CTC diagnostic technology for clinical use and the only actionable test for detecting CTCs in cancer
patients with metastatic breast, prostate or colorectal cancer. This device is already being applied in
clinical studies [241]. However, with the high technology advances that are being observed from year
to year, the majority of the platforms described in this review may be in a near future optimized to fit
FDA rules.
Comparing the inherent limitations of BoCs with the limitations existent in in vitro models and
with the complexity found in animal models, BoC devices represent a step forward. Indeed, BoCs
play a role as gateways for a comprehensive platform, which allows identifying multi-organ toxicity
and/or decreased efficacy due to metabolic activity. BoCs have the ability to not only improve the

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drug development process significantly, but also to improve the knowledge on tissue-tissue and/or
tissue-biomaterials interactions, diminishing the gap between in vivo and in vitro conditions in tissue
engineering applications and disease progression studies.

5. Concluding Remarks
The potential of microfluidics to fuel research applications and enter routine clinical practice is
indeed impressive. The use of microfluidic platforms as biomedical tools holds the promise to be
further implemented as clinically-relevant devices to be included in the daily healthcare practice,
by anticipating and monitoring the onset of diseases. Due to the interplay among microfluidics,
biosensors and tissue engineering know-how, diagnostics is becoming faster and cheaper, and
biomedical devices are getting more comprehensive and able to restore complex lost functions of
diseased or damaged tissues and organs.
In research, microfluidics is used as a complement to several methodologies and prototypes to
solve bench issues or to improve existing technologies. Several publications describe proof-of-concept
devices as innovative and smart alternatives for biomedical applications, although their FDA
approval, standardization and further manufacturing in large-scale is still far to come. FDA rules
are very restrictive in the direct use of microfluidics for tissue engineering applications; however
this technology can be easily adapted to host 3D microtissues and BoC devices, offering better
predictability of drug effects than conventional 2D test systems. These models enable a deep
understanding of interactions between drugs and their metabolites in various organs with regard to
toxic effects and/or drug efficacy. Despite the majority of the biomedical applications of microfluidics
being in vitro or ex vivo, in the near future the use of microfluidic devices will most likely be preferred
to in vivo studies, and upscaled to be suitable for the diagnostics and clinical scenarios, like in the
capture of circulating tumor cells and clusters.
Microfluidic technology is thus deemed to have a huge impact on science and medicine practice
due to its rapid progress, to its tunability and scalability, which leads to outline a trajectory of
tremendous innovation with countless potential. From simple to complex systems, microfluidics will
be evolving, being part of breakthrough and futurist ideas, thus playing a role in the improvement of
organ-on-a chip studies to body-on-a-chip approaches.

Acknowledgments: The present work was supported by the European Regional Development Fund-Project
FNUSA-ICRC (No. CZ.1.05/1.1.00/02.0123). A.C.P.Águas is a F.C.T. doctoral fellow (SFRH/BD/88958/2012).
This work was supported by Fundação para a Ciência e a Tecnologia (FCT), Portugal (UID/BIM/04773/2013).
A.R. is supported by the Internal Research Grant Program, Università Campus Bio-Medico di Roma. We
acknowledge Victorio Pozo Devoto, Rui Borges dos Santos and Carina Silva proof reading and scientific
discussion and Dr. Jorge Oliver de la Cruz for support in Adobe Illustrator software.
Conflicts of Interest: The authors declare no conflict of interest.

List of Abbreviations
2D—two-dimensional
3D—three-dimensional
3MD—Three-dimensional microfluidic microanalytical micro-organ device
ADME—absorption, distribution, metabolism, and excretion
BBB—blood-brain barrier
BioMEMS—biological/biomedical micro electro mechanical systems
BOC—body-on-a-chip
COPD—chronic obstructive pulmonary disease
CTCs—circulating tumor cells
ECM—extracellular matrix
GEDI—geometrically enhanced differential immunocapture
GIT—gastrointestinal tract

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HGF—hepatocyte growth factor

IDCCM—integrated dynamic cell culture microchip
IIDMP—integrated insert in a dynamic microfluidic platform
LOC—lab-on-a-chip
MMD—Microfluidic-multilayer device
OoC—organ-on-a-chip
PDMS—poly(dimethylsiloxide)
PEG—poly(ethylene glycol)
PIPAAm—poly(N-isopropylacrylamide)
PMMA—poly(methyl methacrylate)
PTFE—poly(tetrafluoroethylene)
RWG—resonant waveguide grating
SAW—surface acoustic wave
TE—tissue engineering
UPLC-IM-MS—ultra-performance liquid chromatography-ion mobility-mass
µBVM—microscale blood vessel module
µCCA—microscale cell culture analog

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