Wu 

et al. BioMed Eng OnLine (2020) 19:9

[Link] BioMedical Engineering
OnLine

REVIEW Open Access

Organ‑on‑a‑chip: recent breakthroughs

and future prospects
Qirui Wu1, Jinfeng Liu1, Xiaohong Wang1, Lingyan Feng1, Jinbo Wu1, Xiaoli Zhu2, Weijia Wen1
and Xiuqing Gong1* 

*Correspondence:
gongxiuqing@[Link] Abstract 
1
Materials Genome Institute, The organ-on-a-chip (OOAC) is in the list of top 10 emerging technologies and refers to
Shanghai University,
Shanghai 200444, China a physiological organ biomimetic system built on a microfluidic chip. Through a combi-
Full list of author information nation of cell biology, engineering, and biomaterial technology, the microenvironment
is available at the end of the of the chip simulates that of the organ in terms of tissue interfaces and mechanical
article
stimulation. This reflects the structural and functional characteristics of human tissue
and can predict response to an array of stimuli including drug responses and envi-
ronmental effects. OOAC has broad applications in precision medicine and biological
defense strategies. Here, we introduce the concepts of OOAC and review its application
to the construction of physiological models, drug development, and toxicology from
the perspective of different organs. We further discuss existing challenges and provide
future perspectives for its application.
Keywords:  Organ-on-a-chip, Microfluidic chip, Physiological model, Human organs,
Stem cell

Background
Microfluidics is a science and technology that precisely manipulates and processes
microscale fluids. It is commonly used to precisely control microfluidic (­ 10−9 to ­10−18 L)
fluids using channels that range in size from tens to hundreds of microns and is known
as a “lab-on-a-chip” [1–4]. The microchannel is small, but has a large surface area and
high mass transfer, favoring its use in microfluidic technology applications including
low regent usage, controllable volumes, fast mixing speeds, rapid responses, and preci-
sion control of physical and chemical properties [1, 5, 6]. Microfluidics integrate sample
preparation, reactions, separation, detection, and basic operating units such as cell cul-
ture, sorting and cell lysis [7]. For these reasons, interest in OOAC has intensified [8].
OOAC combines a range of chemical, biological and material science disciplines [9] and
was selected as one of the “Top Ten Emerging Technologies” in the World Economic
Forum [10].
OOAC is a biomimetic system that can mimic the environment of a physiological
organ, with the ability to regulate key parameters including concentration gradients
[11], shear force [12], cell patterning [13], tissue-boundaries, [14] and tissue–organ

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Wu et al. BioMed Eng OnLine (2020) 19:9 Page 2 of 19

interactions [15]. The major goal of OOAC is to simulate the physiological environment
of human organs [16].
Human physiology is the science of studying the functions of the human body and its
organ systems. This is of great significance to our understanding of the dysfunction and
pathogenesis of the body, and therefore closely aligns with the fields of medicine, drug
development and toxicology [17]. The most relevant and direct methods for studying
human physiology are in vivo experiments that study human or model organisms. Bod-
ily functions rely on the interaction and adaptation of many lower-level components
such as tissues, cells, proteins and genes. It is therefore challenging to reveal the under-
lying mechanisms of physiological phenomena simply through in  vivo studies [18]. In
addition, drug development and toxicology require the assessment of the physiological
effects of thousands of compounds [19]. Due to the limitations of low-throughput in vivo
testing, biologists use in vitro cell culture. Cell culture refers to the growth and mainte-
nance of cells in a controlled environment [20]. For decades, traditional two-dimensional
(2D) cell culture systems formed an important platform for life science research. Using
2D systems, the functions of various cells are studied by culturing cells or cell products.
However, 2D systems fail to accurately simulate the physiological manifestations of liv-
ing tissues/organs, intra-organ interactions and microenvironmental factors [21, 22] and
often require verification in in  vivo animal models. Due to species differences, animal
experiments often fail to replicate human experiments [23], and due to both high costs
and ethical issues, the use of animals as models for drug testing has come under scrutiny
[24]. In preclinical testing, an inadequate description of the human tissue environment
may lead to inaccurate predictions of the combined effects of overall tissue function [25].
OOAC was designed to overcome these shortcomings by providing more physiological
model systems [26]. OOAC was proposed as a future replacement technology for experi-
mental animal models [27].
This review introduces recent advances from OOAC technology and discusses its
future perspectives for cell biological assessments.

Organs‑on‑a‑chip design concept and key components

Design concept
Culture systems require the control of external and internal cell environments [28].
OOAC combined with micromachining and cell biology can control external parameters
and accurately simulate physiological environments [16]. Dynamic mechanical stress,
fluid shear and concentration gradients are required on the chip. Cell patterning should
also be realized to fully reflect physiological processes.

Fluid shear force  Microfluidics enables the dynamic culture of cells through micro-
pump perfusion, which facilitates the administration of nutrients and timely waste dis-
charge. The dynamic environment in which cells are located is more comparable to in vivo
conditions than static culture. In addition, fluid shear stress induces organ polarity [29].
Importantly, OOAC exerts necessary physical pressure on the normal biological func-
tions of endothelial cells [30] by activating cell surface molecules and associated signaling
cascades. Similarly, the incorporation of fluid into the OOAC device permits biological
assessments at the single organ level [31]. The OOAC system summarizes flow through a
 Wu et al. BioMed Eng OnLine (2020) 19:9 Page 3 of 19

simple “rocker” on a chip fluid motion, or through a more complex programmable “pulsa-
tile” format, arranged in a single loop for organization-specific configurations [32].

Concentration gradient  At the microscale level, the fluid acts primarily as a laminar
flow, resulting in a stable gradient of biochemical molecules, controlled both spatially
and temporally. Various biochemical signals driven by concentration gradients exist in
biological phenomena, including angiogenesis, invasion, and migration [33–35]. Micro-
fluidics simulate complex physiological processes in the human body by altering flow
velocity and channel geometry using microvalves and micro-pumps to achieve stable,
three-dimensional (3D) biochemical concentration gradients.

Dynamic mechanical stress  Normal day-to-day organ pressure includes blood pres-
sure, lung pressure, and bone pressure. These pressures play a major role in maintaining
mechanically stressed tissues such as skeletal muscle, bone, cartilage and blood vessels
[36–38]. Microfluidics enable the use of elastic porous membranes to create periodic
mechanical stresses. This mechanical stimulation is considered a key determinant of dif-
ferentiation during physiological processes [39, 40].

Cell patterning  The organization of the human body requires a complex and ordered
arrangement of multiple cells to form a functional whole body interactions. Microfluidics
control cell patterning for the construction of in vitro physiological models with complex
geometries. Surface modifications [41], templates [42], and 3D printing [43] contribute
to cell patterning on the chip. The 3D printing method enables multi-scale cell patterning
by permitting the formation of hydrogel scaffolds with complex channels. The advantage
of 3D printing is to allow user-defined digital masks to provide versatility in cell pat-
terns, critical for the in vitro reconstruction of the cellular microenvironment. Li et al.
[44] developed methods to achieve rapid heterotypic cell patterning on glass chips using
controlled topological manipulations. This method combines a polyvinyl acetate coating,
carbon dioxide laser ablation, and continuous cell seeding techniques on a glass chip. This
method enables controlled epithelial–mesenchymal interactions. In addition, mesenchy-
mal cells with similar properties can also be patterned on glass chips. This method can be
helpful for large-scale investigation and pharmaceutical testing of cutaneous epithelial–
mesenchymal interaction and can also be applied to the patterning of other cells.

Key components
The OOAC involves four key components, including (1) microfluidics; (2) living cell tis-
sues; (3) stimulation or drug delivery; and (4) sensing [45]. The microfluidic component
refers to the use of microfluidics to deliver target cells to a pre-designated location and
includes a system of culture fluid input and waste liquid discharge during the culture
process. Typically, this component is characterized by miniaturization, integration, and
automation [7]. The living cell tissue component refers to components that spatially
align a particular cell type in the case of 2D or 3D systems. The 3D arrangements are
typically created by the addition of biocompatible materials such as hydrogels. These
materials can prevent mechanical damage and shape three-dimensional arrangements.
[42]. Although the 3D tissue structure more accurately simulates the in  vivo situation
Wu et al. BioMed Eng OnLine (2020) 19:9 Page 4 of 19

compared to 2D models, due to the limitations of technology and cost and the assem-
bly of extracellular matrix and the presetting and formation of vasculature, living cell in
organ tissues are still mostly cultivated in 2D. For certain tissues, physical or chemical
signals are required to simulate the physiological microenvironment, which promotes
micro-tissue maturation and function. For example, electrical stimulation can help
myocardial tissue maturation [46]. Different signal stimuli can be derived from for drug
screening approaches [47]. The sensing component for detecting and compiling data
can be an embedded sensing output component or a transparent chip based visual func-
tion evaluation system. Peel et  al. [48] used automated systems to image multicellular
OOACs, producing detailed cell phenotypes and statistical models for measurements.
Kane et  al. [49] developed a cell system to monitor cells in a 3D microfluidic setting.
These assays featured time-lapse imaging microscopy to assess cellular electrical activity
through quality control. A meaningful human-on-chip cell model cannot be described
and accessed without microsensors-mediated reading of the metabolic state at charac-
teristic points in the system.

Emerging OOAC technologies

Liver OOAC
The hepatic system is the major site of drug/toxin metabolism. The liver constitutes a
series of complex hepatic lobules that confer multicellular functional communication
[50]. Maintaining the physiology of hepatocytes over an extended time period is chal-
lenging [51]. Kane et  al. designed the first liver based system that consisted of micro-
fluidic pores in which 3T3-J2 fibroblasts and rat liver cells were co-cultured to mimic
an airway interface (Fig.  1) [52]. Rat hepatocytes cultured in the chip could continu-
ously and stably synthesize albumin and undergo metabolism. Lee et  al. [53] designed
a chip that mirrored the interstitial structure of endothelial cells and cultured primary
hepatocytes, with culture media perfused outside the gap. This permeable endothelial
gap separated hepatocytes in cord-based structures permitting their separation from the
external sinusoidal region, simultaneously maintaining efficient substance exchange. Ho
et al. [13] used radial electric field gradients that were produced using electrophoresis to
pattern cells onto circular polydimethylsiloxane (PDMS) chips. These novel techniques
simulated the hepatic lobule structure. Hegde et  al. [54] fabricated a 2-layer chip that
separated the channels using a porous polyethylene terephthalate(PET) membrane and
continuously perfused collagen and fibronectin-sanded rat primary hepatocytes into the
lower channel through the upper chamber.
To improve the physiological models, 3D hepatocyte culture techniques have been
used form microfluidic chips [55]. Ma et al. [56] produced a biomimetic platform for
the perfusion of hepatic spheroids in situ. Yum et al. [57] produced systems to study
how hepatocytes affect other cell types. High-throughput assays were developed to
assess liver cell drug toxicity. Riahi et  al. [58] produced microfluidic electrochemi-
cal chip immunosensors to detect the biomarkers produced during hepatotoxicity.
Chong et  al. [59] produced assays to monitor drug skin sensitization through the
assessment of metabolite production and the activation of antigen presenting cells
(APCs). This system holds value as a drug screening platform to identify compounds
that produce systemic skin reactions. Lu et al. [60] developed biomimetic liver tumors
 Wu et al. BioMed Eng OnLine (2020) 19:9 Page 5 of 19

Fig. 1  Schematic of the DLM-based liver tumor-on-a-chip. a Preparation of the DLM solution from a natural
liver; b 3D schematic representation of the various components of the equipment (top and bottom, top and
bottom microchannels, PET membrane, air inlet, and outlet) and their respective dimensions (reprinted with
permission from [52] Copyright © 2006, Royal Society of Chemistry)

through integrating decellularized liver matrixes (DLM) with gelatin methacryloyl

(GelMA) to mirror the 3D tumor microenvironment (TME). This system provides
an improved disease model for a range of future anti-cancer pharmacological stud-
ies. Furthermore, a number of disease or injury states were tested. Kang et  al. [61]
used their system to analyze viral replication of the hepatitis B virus. Zhou et al. [62]
developed a system for modeling alcohol injury. Further characterization of cultured
cytoplasm in metabolomics, proteomics, genomics, and epigenomic analysis will help
improve the functional outcome of these studies.

Lung‑on‑a‑chip
Gas exchange in the lungs is regulated by the alveoli which can be challenging to repro-
duce in vitro. Microfluidics can establish extracorporeal lung models and lung patholo-
gies through accurate fluid flow, and sustained gaseous exchange. Current studies have
focused on the regulation of airway mechanical pressure, the blood–blood barrier (BBB),
[63] and the effects of shear force on pathophysiological processes. Huh et al. produced
a lung-on-a-chip model (Fig. 2) [64] using soft lithography to divide the chip into regions
separated by 10 μm PDMS membranes with an extracellular matrix (ECM). The upper
PDMS regions had alveolar epithelial cells, whilst the lower regions contained human
pulmonary microvascular endothelial cells, thus mimicking the alveolar–capillary bar-
rier. The structures of the membranes were altered under a vacuum to simulate expan-
sion/contraction of the alveoli during respiration. Inflammatory stimuli were introduced
into the system through neutrophils that were passed to the fluid channels. This pro-
duced a pathological model of pulmonary edema through the introduction of interleu-
kin-2 (IL-2) [65]. This highlights the utility of the OOAC models to improve current
in vivo assays.
Wu et al. BioMed Eng OnLine (2020) 19:9 Page 6 of 19

Fig. 2  Lung-on-a-chip system. a An alveolar–capillary barrier was produced on porous flexible PDMS
membranes coated with ECM using spaced PDMS microchannels. The device reproduced respiratory motion
through a vacuum leading to mechanical stretching and the formation of an alveolar–capillary barrier; b
following inhalation, the diaphragm contracts, reducing pleura pressure. The alveolar–capillary interface
became stretched due to alveoli tension; c device development: a porous membrane between the upper
and lower channels bound irreversibly following plasma exposure; d PDMS moved through the side of the
channels and then was removed following vacuum pressure. e Actual images of the device (reprinted with
permission from [64] Copyright © 2010, American Association for the Advancement of Science)

In 2015, Stucki et al. [66] reported a lung chip that mimicked the lung parenchyma.
The system included an alveolar barrier and 3D cyclic strain that mimicked respiration
representing the first elastic membrane expansion model to simulate breathing. Blume
et al. [67] produced 3D airway culture models that simulated pulmonary interstitial flow
through the exchange of both fluid and media. This permitted more in-depth physiologi-
cal studies of the epithelial barrier. This model utilizes a stent with a permeable filter
as a single tissue culture chamber and combined multiple chambers for improved inte-
gration. In the lung-on-a-chip, whilst simulating lung gas–liquid interfaces and respira-
tory dilation through the microfluidic system, pressure can be applied to the alveoli and
attached capillaries, providing a shear flow profile. This realistically simulates the lung
environment. Humayun et  al. [68] cultured airway epithelial and smooth muscle cells
at different sides of a hydrogel membrane to assess their suitability as a physiological
model. The system was combined with microenvironment cues and toxin exposure as a
physiological model of chronic lung disease. Yang et al. [69] produced a poly(lactic-co-
glycolic acid) (PLGA) electrospinning nanofiber membrane as a chip matrix for cell scaf-
folds. Given the ease of the system, it is applicable to lung tumor precision therapy and
tissue engineering approaches was highlighted.
Lung tissue organ chips are useful as implantable respiratory assistance devices. Peng
et al. [70] designed lung assist devices (LAD) to permit additional gas exchange in the
placenta for preterm infants during respiratory failure. The concept of large-diameter
channels was achieved in the umbilical arteries and veins, providing LAD with high
extra-corporeal blood flow. This has added utility because clinical trials for umbilical
vasodilation thresholds were unethical. This study was the first to systematically quan-
tify umbilical vessel damage as the result of expansion by catheters. Dabaghi et al. [71]
performed microfabrication for microfluidic blood oxygenators using double-sided gas
delivery to improve gas exchange. Oxygen uptake increased to 343% in comparison
 Wu et al. BioMed Eng OnLine (2020) 19:9 Page 7 of 19

to single-sided devices. Xu et  al. [72] used a microfluidic chip platform to mimic the
microenvironment of lung cancer with cancer cell lines and primary cancer cells and
tested different chemotherapeutic drugs. Another recent study mimicked asthma in a
“small airway-on-a-chip” model [73]. With the models of human asthmatic and chronic
obstructive pulmonary disease airways, therapeutics were tested and the chip model
recapitulated in vivo responses to a similar therapy.

Kidney OOAC
The kidney is responsible for the maintenance of osmotic pressure drug excretion. Kid-
ney toxicity leads to an irreversible loss of renal filtration highlighting the need for drug
screening systems. Filtration and reabsorption take place in the nephrons that consist
of the glomerulus, renal capsule, and renal tubule. Microfluidics can simulate the fluid
environment that support tubular cell growth, and provides porous membrane support
for the maintenance of cell polarity [16].
Jang et al. [74] produced the first multi-layered microfluidic system (Fig. 3a) in which
mouse kidney medullary collecting duct cells were used to simulate renal filtration. The
device provided a biomimetic environment that enhanced polarity of the inner medul-
lary collecting duct through promoting cytoskeletal reorganization and molecular trans-
port in response to hormone stimulation. In 2013, the same microfluidic device was
used to culture human primary renal epithelial cells [75]. These were the first toxicity
studies of primary kidney cells. This device enables direct visualization and quantitative
analysis of diverse biological processes of the intact kidney tubule in ways that have not

Fig. 3  a Kidney tubular chip. Sandwich assembly of the PDMS channel, porous membrane, and PDMS
reservoir (reproduced from [74]); b the channel can replicate the urinary cavity and capillary lumen of the
glomerulus. The porous flexible PDMS membrane can be used to functionalize the protein laminin to mimic
the glomerular basement membrane. Cyclic mechanical pressure to the cell layer via vacuum stretching of
the flexible PDMS film can be produced (reprinted with permission from [76] Copyright © 2018, Royal Society
of Chemistry)
Wu et al. BioMed Eng OnLine (2020) 19:9 Page 8 of 19

been possible in traditional cell culture or animal models, and it may also prove to be
helpful for studying the basic molecular mechanisms of kidney function and disease.
The disadvantage of conventional cell culture systems is that cell differentiation into
functional cells requires extended culture times and an external signal detection system.
Musah et al. [76] described methods to induce pluripotent stem cell-derived podocytes
to form human glomerular chips (Fig. 3b) in organ culture devices. These mimicked the
structure and function of the glomerular capillary wall, which was not possible with
previously employed methods. The chip was applicable for nephrotoxicity assessments,
therapeutic development, regenerative medicine, and kidney development and disease.
Sakolish et al. [77] produced a reusable microfluidic chip in human proximal tubules and
glomeruli that permitted renal epithelial cells to grow under various conditions. Shear
stress causes nephrotoxicity. Schutgens et  al. [78] designed stable tubule culture sys-
tems that permitted extended expansion and human kidney tissue analysis. Based the
system, a multi-purpose primary renal epithelial cell culture model was developed that
enabled rapid and individualized molecular and cellular analysis, disease modeling, and
drug screening. Tao et  al. [79] presented a powerful strategy to generate human islet
organoids from human induced pluripotent stem cells. This strategy was applicable to a
range of applications for stem cell-based organic engineering and regenerative medicine.

Heart‑on‑a‑chip
Cardiovascular deaths are the leading cause of human mortality. The emergence of
microfluidics has enabled in  vitro bionic studies of cardiac tissue. The myocardium is
a major component of the heart. The beating of cardiomyocytes (CMs) can be used
to directly assess drug effects and is directly related to heart pumping [80]. In 2012,
Grosberg et  al. [81] used PDMS to produce an elastic film with a surface texture and
implanted neonatal rat CMs on the membrane to form a muscle membranes. As the
CMs contract, the muscle film curled to one side. By measuring the degree of this curl
it was possible to analyze the differences in the size of the cell contractile capacities on
the PDMS film. The experimental system was suitable for both single muscle membrane
measurements and high-throughput automated multi-plate assays. Subsequently, in
2013, Zhang et al. [82] utilized hydrogels to produce self-assembled myocardial sheets
in a PDMS model. The CMs were derived from differentiated myocardium. Micro-organ
tissue chips were produced from 3D printing technology that permitted the integration
of myocardial and vascular systems [83]. The model utilized vascular endothelial cells
to form vascular networks and CMs were added to the vascular network gap. The organ
chip produced a screening platform for CV-related drugs.
Zhang et al. [84] introduced the heart-on-a-chip device that used high-speed imped-
ance detection to assess cardiac drug efficacy. The device records the contraction of
CMs to reveal drug effects. The chip represented a preclinical assessment of drug car-
diac efficacy. Marsano et al. [85] built a heart organ platform (Fig. 4) that mimicked the
physiological and mechanical environment of CMs. Direct visualization and quantitative
analysis was performed, which was not permitted in traditional cell culture or animal
models. This platform represents an advance in the field and provides standard func-
tional 3D heart models. This makes the device an innovative and low-cost screening
platform to improve the predictive power of in  vitro models. Schneider [86] designed
 Wu et al. BioMed Eng OnLine (2020) 19:9 Page 9 of 19

Fig. 4  3D heart-on-a-chip. a Two separate PDMS microchambers were employed. The CMs are positioned
in the central channel to create a 3D construct, whilst the medium is replaced trough side-channels; b
the lower end of the compartment is pressurized to deform the PDMS membrane and compress the 3D
structure. Compression is converted to uniaxial strains applied to the 3D cell structure; c PDMS layers are
aligned and irreversibly combined. Upper layers are present in the culture chamber and the drive chambers
represent the lower layers; d 3D illustration; e real-life chip; f SEM of the chip cross section (reprinted with
permission from [85] Copyright © 2016, Royal Society of Chemistry)

convenient and efficient chips to generate heart tissue in a controlled environment based
on human induced pluripotent stem cells. The viability and function of myocardial tis-
sue was maintained for an extended time period and detailed spatiotemporal pulsation
dynamics were optically detected. This platform can be used for a variety of biomedical
applications. In addition, Tzatzalos et al. [87] reported that the hiPSC-CMs can repre-
sent an unlimited potential for healthy and disease-specific CMs to assess the efficacy of
drugs for dilated cardiomyopathy. These advances in drug development have important
implications for cardiovascular tissue because cardiotoxicity is often seen in drug trials
and is one of the main reasons clinical trials are suspended or drugs are withdrawn from
the market.

Intestine‑on‑a‑chip
Oral drugs have to transverse the small intestine to enter the bloodstream. Villi are
key to absorption and their morphology must be maintained on the chip [88]. Imura
et al. [89] developed chips to simulate the intestinal system, consisting of a glass slide
permeable membrane and PDMS sheet containing the channels. Caco-2 cells were
cultured on the chips. Sung et al. produced the first 3D hydrogel structure to simulate
Wu et al. BioMed Eng OnLine (2020) 19:9 Page 10 of 19

the human intestinal villi [90]. Kim et  al. produced bionic devices (Fig.  5) [91]. The
microenvironment of the intestine was reconstructed through shear force and cyclic
strains. Caco-2 cells show prolonged growth and maintained the microbial flora in
the human intestine. The complex structure and physiology of the intestine provided
a platform for drug screening and the role of the intestinal microbiome, inflammatory
cells and peristaltic-related mechanical deformation during intestinal disease [92].
The device permitted the exploration of the etiology of intestinal disease and identi-
fied therapeutic targets and drugs. This study demonstrates the potential of intestine-
on-chip for personalized medicine studies on intestinal cells.
Intestinal cells were cultured alone or with endothelial cells including HUVECs
[91]. Genome fidelity was low, so the chips mimicked intestinal function. Kasen-
dra1 et al. [93] combined intestinal tissue engineering [94] and OOAC technology to
establish in vitro biological models of the human duodenum. The intestinal epithelial
cells cultured in the chip were obtained from endoscopic biopsies or organ resections.
This chip represented the closest model to the living duodenum and reproduced key
features of the small intestine. Recent findings enhanced our knowledge of the intesti-
nal microbiome [95] and intestinal morphology [96].

Fig. 5  a Illustration of the intestine-on-a-chip device; b images of the device composed of transparent PDMS
elastomers; c cross-sectional view of the channels and square illustrations showing a top view of the porous
film; d schematic of intestinal monolayers cultured on the chips (top) and phase contrast images (bottom)
plus (left) or minus (right) mechanical strains (30%); arrows indicate the direction). e pressure quantitation
(reprinted with permission from [91] Copyright © 2012, Royal Society of Chemistry)
 Wu et al. BioMed Eng OnLine (2020) 19:9 Page 11 of 19

Multi‑organs‑on‑a‑chip
An array of physiological pathways requires continuous media circulation and inter-
tissue interactions. Single organ chips fail to fully reflect the complexity, functional
changes, and integrity of organ function [97]. The “multi-organ-on-a-chip”, otherwise
referred to as the “human-on-a-chip” [98] simultaneously constructs multiple organs
attracting obvious research attention. Multi-organs-on-a-chip culture cells of different
organs and tissues simultaneously which are connected by channels (bionic blood vessel
[99]), to achieve multi-organ integration, permitting the examination of interactions to
establish a system [100, 101]. These can be separated into static, semi-static and flexible
approaches [102]. Static multiple organs are integrated into single connected devices. In
semi-static systems, the organs are joined via fluidic networks with T ­ ranswell®-based
[103] tissue inserts. In the flexible system, individual organ-specific platforms are inter-
connected using flexible microchannels. In such systems, the flexible nature is advan-
tageous and recreates multiple organs [102]. Although the multi-organs-on-a-chip
concept remains in its infancy, major breakthroughs have been made, including the
design of two-organs [104, 105], three-organs [106, 107], four-organs [108, 109], and ten
organs on the chip [110].
In 2010, Van et al. [104] were the first to combine liver and intestines in a microfluidic
device. The intestine and liver slices functioned on the chip and demonstrated its appli-
cability to organ interactions including the regulation of bile acid synthesis. This system
enabled in  vitro studies and provided insight into organ–organ interactions. A larger
number of organs have since been concentrated onto individual chips. Organ chips are
required to maintain stable fluid connection, avoid bacterial contamination, and moni-
tor cell viability throughout the culture process. As the number of organs on the chip
increases, the complexity of the system is enhanced, inevitably leading to unpredictable
results. Simplifying existing systems is critical to achieving a wider range of applications.
Lee et  al. [111] fabricated pumpless, user-friendly multi-organs-on-a-chip which were
easily assembled and operated. Satoh et  al. reported a multi-throughput multi-organ-
on-a-chip system formed on a pneumatic pressure-driven medium circulation platform
that was microplate-sized (Fig. 6) [112]. This system possesses the following advantages
for application to drug discovery: simultaneous operation of multiple multi-organ cul-
ture units, design flexibility of the microfluidic network, a pipette-friendly liquid han-
dling interface, and applicability to experimental protocols and analytical methods
widely used in microplates. This multi-organ culture platform will be an advantageous
research tool for drug discovery.
The continued development of OOAC was dependent on advances in design, mode-
ling, manufacturability, and usability. Lantada et al. [113] produced an innovative combi-
nation of laser technologies. The assessment of human mesenchymal stem cells verified
the effectiveness of the technique and the resultant chip was transparent, facilitating
imaging procedures. Such technologies are feasible for mass-produced chips and hold
utility for energy, transportation and aerospace industries.
OOAC technology has developed rapidly in recent years and has enhanced our knowl-
edge of all the major organs. Others not discussed in this review include blood vessels
[99, 114, 115], the skin [116, 117], the BBB [118, 119], skeletal muscle [120, 121], and the
CNS [122, 123].
Wu et al. BioMed Eng OnLine (2020) 19:9 Page 12 of 19

Fig. 6  a Multi-throughput multi-organ-on-a-plate systems; b projection of a culture device containing a

4 × 4 culture chamber illustrated through a culture chamber of an X–X’ cross section; c design of microfluidic
networks in the microfluidic plates for 8-throughput 2-organ systems and a 4-throughput 4-organ system.
Design of the microfluidic networks in microfluidic plates for eight-channel dual-organ systems and
four-flux four-organ systems. Closed circles indicate the location of the hole leading to the top surface of the
microfluidic plate. Dark and light-shaded areas are deep and shallow microfluidic channels, respectively. Areas
surrounded by green lines represent the circulation culture unit. Blue lines indicate the wall of the culture
room. Thin red lines surrounding the exit indicate the Laplace valve. d Media circulation was performed using
pneumatic pressure in the two-organ system. Red arrows indicate the direction of media flow (reprinted with
permission from [112] Copyright © 2017, Royal Society of Chemistry)

Stem cell engineering

The source of biological tissue is one of the most important parameters in OOAC
design. Stem cells can be extracted from humans without tissue biopsy [124]. By defini-
tion, a stem cell is any cell that is self-renewing and has the potential to differentiate into
one or more specialized cell types. The most common types include embryonic stem
cells (ESCs), induced pluripotent stem cells (iPSCs), and adult stem cells (ASCs). These
cells can be used as a biological tissue source for OOAC (Fig. 7) [125]. The most com-
mon human ASCs are mesenchymal stem cells (MSCs) which are pluripotent stem cells
extracted from adult tissue [126]. Bone marrow mesenchymal stem (bMSCs) cells are
typically derived from bone marrow or adipose tissue, making them an attractive option
due to their ease of extraction from tissue biopsies [127]. Due to their limited ability
to differentiate, lack of consistent derivation protocols and clear biological responses,
MSCs are less useful in OOAC models than their pluripotent counterparts. Human ESCs
originate from blastocysts or internal cells of the embryo. Dependent on the source, they
can be pluripotent and differentiate into any type of adult cell from any of the three germ
layers [128]. However, human ESCs must be derived from human embryos which is eth-
ically controversial, in turn leading to regulations and restrictions. Due to the ethical
debate surrounding ESCs and the technical difficulties of producing large numbers of
genetically diverse cell lines, it is more difficult to apply human ESCs to clinical trials
than their use as precision drug replacements in disease models for therapeutic drug
evaluation [129]. Like ESCs, MSCs are pluripotent and can differentiate from all three
 Wu et al. BioMed Eng OnLine (2020) 19:9 Page 13 of 19

Fig. 7  Tissue sources for the organ-on-a-chip (OOAC) devices. Embryonic stem cells (ESCs), induced
pluripotent stem cells (iPSCs), and adult stem cells (ASCs) can be differentiated and integrated into
microfluidic chips as for cell lines and primary cells. The figure illustrates the advantages (white) and
limitations (black) of ESCs, ASCs, iPSCs, primordial and tissue biopsies, and cell lines in OOC devices. Cell
lines and primary cells are more common in oocytes as they typically display good biological response
characteristics. However, cell lines do not represent normal physiological conditions and primary cell culture
time is limited, and the quality is unstable. In contrast, stem cells are readily available and are an infinite
cell source. Even with current limitations on differentiation and maturation protocols, stem cells represent
a promising technology that can be incorporated into OOC devices (reprinted with permission from [125]
Copyright © 2019, Elsevier)

germ layers [130]. As iPSCs are derived from adult tissue rather than embryonic tissue,
they avoid the ethical issues associated with ESCs. No significant differences in gene
expression levels, surface marker expression, and morphology between ESCs and iPSCs
are observed in cells from the same genetic background [131, 132]. In addition to cir-
cumventing ethical controversies, another advantage of iPSCs over ESCs is that they can
be obtained from donors of known disease phenotypes, which can be used for patient-
specific disease models and drug screening.
Since stem cells are more readily available than many primitive cell types and tis-
sue biopsies, and they are more physiologically representative than other cell lines and
are likely to become the main tissue source for future OOAC (Fig. 8) [133]. Continued
research into the methods by which stem cells differentiate into functional organ models
on chips will contribute to improvements in stem cell methods and advances in OOAC
technology [125, 134].

Conclusion and future perspectives

We have reviewed recent progress in OOAC technology. Microfluidic chips provide
favorable support for the development of OOAC. Its development has attracted world-
wide research attention and great scientific advances have been made. A large number of
OOACs have been designed and prepared. An array of human organs has been studied.
The ultimate goal of OOAC is to integrate numerous organs into a single chip, and to
build a more complex multi-organ chip model, finally achieving a “Human-on-a-chip”.
Wu et al. BioMed Eng OnLine (2020) 19:9 Page 14 of 19

Fig. 8  Future trends in stem cell research. a Building blocks. b Organ-on-a-chip techniques can mimic
real-life in vivo states (reprinted with permission from [133] Copyright © 2015, John Wiley and Sons)

Although OOAC technology has developed rapidly, the human-on-a-chip theory

remains distant. PDMS is the most widely employed material, but comes with dis-
advantages as the resultant film is thicker than the in vivo morphology. A decreased
absorbance of small hydrophobic molecules influences solvent efficacy and toxicity.
It is thus necessary to identify suitable alternative materials. At present, the cost of
manufacturing and experimental implementation is relatively expensive, which is not
conducive to the widespread use of organ chips, so components must be of low cost
and easy to dispose. More expensive components should be reusable. In terms of inte-
grated system components, the media volume and connector size must be reduced for
general use. Collecting samples on the chip may interfere with its operation, result-
ing in changes in the concentration of various metabolites. More suitable sensors are
thus required. Universal cell culture mediums suitable for all organs are also required.
Most critically, as the number of organs on the chip increases, functionality becomes
more complex and generated data carry artefactual and non-translatable risks. This
is currently unsolvable. In the case of long-term repeated administration or on-chip
studies, the biomarkers identified in vitro may not fully reflect the in vivo equivalent.

Abbreviations
OOAC: Organ-on-a-chip; 2D: Two-dimensional; 3D: Three-dimensional; PDMS: Polydimethylsiloxane; PET: Polyethylene
terephthalate; APCs: Antigen presenting cells; DLM: Decellularized liver matrixes; GelMA: Gelatin methacryloyl; TME:
Tumor microenvironment; BBB: Blood-blood barrier; ECM: Extracellular matrix; IL-2: Interleukin-2; PLGA: Poly(lactic-co-
glycolic acid); LAD: Lung assist devices; CMs: Cardiomyocytes; ESCs: Embryonic stem cells; IPSCs: Induced pluripotent
stem cells; ASCs: Adult stem cells; BMSCs: Bone marrow mesenchymal stem.

Acknowledgements
Not applicable.

Authors’ contributions
QW conceived and wrote the review. JL, XW, LF, JW, XZ, WW, and XG contributed to writing and critically reviewed the
manuscript. All authors read and approved the final manuscript.

Funding
This work was sponsored by the Shanghai Pujiang Program (17PJ1402800) and the National Natural Science Foundation
of China (21775101).

Availability of data and materials

Not applicable.
 Wu et al. BioMed Eng OnLine (2020) 19:9 Page 15 of 19

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests
The authors declare that they have no competing interests.

Author details
1
 Materials Genome Institute, Shanghai University, Shanghai 200444, China. 2 School of Life Sciences, Shanghai University,
Shanghai 200444, China.

Received: 11 November 2019 Accepted: 5 February 2020

References
1. Whitesides GM. The origins and the future of microfluidics. Nature. 2006;442:368–73. https​://[Link]/10.1038/natur​
e0505​8.
2. Squires TM, Quake SR. Microfluidics: fluid physics at the nanoliter scale. Rev Mod Phys. 2005;77:977–1026. https​://
[Link]/10.1103/RevMo​dPhys​.77.977.
3. Daw R, Finkelstein J. Lab on a chip. Nature. 2006;442:367.
4. Mitchell P. Microfluidics–downsizing large-scale biology. Nat Biotechnol. 2001;19:717–21.
5. Figeys D, Pinto D. Lab-on-a-chip: a revolution in biological and medical sciences. Anal Chem. 2000;72:330A.
6. Haeberle S, Zengerle R. Microfluidic platforms for lab-on-a-chip applications. Lab Chip. 2007;7:1094–110.
7. Kwon J-S, Oh J. Microfluidic technology for cell manipulation. Appl Sci. 2018;8:992. https​://[Link]/10.3390/app80​
60992​.
8. Sosa-Hernández JE, Villalba-Rodríguez AM, Romero-Castillo KD, Aguilar-Aguila-Isaías MA, García-Reyes IE,
Hernández-Antonio A, Ahmed I, Sharma A, Parra-Saldívar R, Iqbal HMN. Organs-on-a-chip module: a review from
the development and applications perspective. Micromachines (Basel). 2018. https​://[Link]/10.3390/mi910​0536.
9. Ahmed I, Akram Z, Bule M, Iqbal H. Advancements and potential applications of microfluidic approaches—a
review. Chemosensors. 2018;6:46. https​://[Link]/10.3390/chemo​senso​rs604​0046.
10. Top Ten Emerging Technologies. 2016. https​://[Link]​[Link]/agend​a/2016/06/top-10-emerg​ing-techn​ologi​
es-2016/.
11. Wang L, Liu W, Wang Y, Wang JC, Tu Q, Liu R, Wang J. Construction of oxygen and chemical concentration gradi-
ents in a single microfluidic device for studying tumor cell-drug interactions in a dynamic hypoxia microenviron-
ment. Lab Chip. 2013;13:695–705.
12. Galie PA, Nguyen DHT, Choi CK, Cohen DM, Janmey PA, Chen CS. Fluid shear stress threshold regulates angiogenic
sprouting. Proc Natl Acad Sci USA. 2014;111:7968–73.
13. Ho CT, Lin RZ, Chen RJ, Chin CK, Gong SE, Chang HY, Peng HL, Hsu L, Yew TR, Chang SF. Liver-cell patterning lab
chip: mimicking the morphology of liver lobule tissue. Lab Chip. 2013;13:3578–87.
14. Booth R, Kim H. Characterization of a microfluidic in vitro model of the blood-brain barrier (μBBB). Lab Chip.
2012;12:1784–92. https​://[Link]/10.1039/c2lc4​0094d​.
15. Sung JH, Shuler ML. A micro cell culture analog (microCCA) with 3-D hydrogel culture of multiple cell lines
to assess metabolism-dependent cytotoxicity of anti-cancer drugs. Lab Chip. 2009;9:1385–94. https​://doi.
org/10.1039/b9013​77f.
16. Bhatia SN, Ingber DE. Microfluidic organs-on-chips. Nat Biotechnol. 2014;32:760–72.
17. Heylman C, Sobrino A, Shirure VS, Hughes CC, George SC. A strategy for integrating essential three-dimensional
microphysiological systems of human organs for realistic anticancer drug screening. Exp Biol Med (Maywood).
2014;239:1240–54. https​://[Link]/10.1177/15353​70214​52529​5.
18. Kieninger J, Weltin A, Flamm H, Urban GA. Microsensor systems for cell metabolism—from 2D culture to organ-
on-chip. Lab Chip. 2018;18:1274–91. https​://[Link]/10.1039/c7lc0​0942a​.
19. Halldorsson S, Lucumi E, Gómez-Sjöberg R, Fleming RMT. Advantages and challenges of microfluidic cell culture in
polydimethylsiloxane devices. Biosens Bioelectron. 2015;63:218–31. https​://[Link]/10.1016/[Link].2014.07.029.
20. Paguirigan AL, Beebe DJ. Microfluidics meet cell biology: bridging the gap by validation and application of micro-
scale techniques for cell biological assays. BioEssays. 2008;30:811–21.
21. Sung JH, Esch MB, Prot J-M, Long CJ, Smith A, Hickman JJ, Shuler ML. Microfabricated mammalian organ sys-
tems and their integration into models of whole animals and humans. Lab Chip. 2013;13:1201–12. https​://doi.
org/10.1039/c3lc4​1017j​.
22. Jiang K, Dong C, Xu Y, Wang L. Microfluidic-based biomimetic models for life science research. RSC Adv.
2016;6:26863–73. https​://[Link]/10.1039/C6RA0​5691A​.
23. van der Meer AD, van den Berg A. Organs-on-chips: breaking the in vitro impasse. Integr Biol (Camb). 2012;4:461–
70. https​://[Link]/10.1039/c2ib0​0176d​.
24. Al-Lamki RS, Bradley JR, Pober JS. Human organ culture: updating the approach to bridge the gap from in vitro to
in vivo in inflammation, cancer, and stem cell biology. Front. Med. (Lausanne). 2017;4:148. https​://[Link]/10.3389/
fmed.2017.00148​.
25. Alépée N. State-of-the-art of 3D cultures (organs-on-a-chip) in safety testing and pathophysiology. Altex. 2014.
https​://[Link]/10.14573​/altex​14061​11.
Wu et al. BioMed Eng OnLine (2020) 19:9 Page 16 of 19

26. Lee SH, Jun B-H. Advances in dynamic microphysiological organ-on-a-chip: design principle and its biomedical
application. J Ind Eng Chem. 2019;71:65–77. https​://[Link]/10.1016/[Link].2018.11.041.
27. Reardon S. ‘Organs-on-chips’ go mainstream. Nature. 2015;523:266. https​://[Link]/10.1038/52326​6a.
28. Young EWK, Beebe DJ. Fundamentals of microfluidic cell culture in controlled microenvironments. Chem Soc Rev.
2010;39:1036–48.
29. Theobald J, Ghanem A, Wallisch P, Banaeiyan AA, Andradenavarro MA, Taskova K, Haltmeier M, Kurtz A, Becker H,
Reuter S. Liver-kidney-on-chip to study toxicity of drug metabolites. ACS Biomater Sci Eng. 2018;4(1):78–89.
30. Davies PF. Flow-mediated endothelial mechanotransduction. Physiol Rev. 1995;75:519–60.
31. Haddrick M, Simpson PB. Organ-on-a-chip technology: turning its potential for clinical benefit into reality. Drug
Discov Today. 2019;24:1217–23. https​://[Link]/10.1016/[Link]​s.2019.03.011.
32. Ronaldsonbouchard K, Vunjaknovakovic G. Organs-on-a-chip: a fast track for engineered human tissues in drug
development. Cell Stem Cell. 2018;22:310–24.
33. Yang KS, Cheng YC, Jeng MS, Chien KH, Shyu JC. An experimental investigation of micro pulsating heat pipes.
Micromachines (Basel). 2014;5:869–72.
34. Nguyen DHT, Stapleton SC, Yang MT, Cha SS, Choi CK, Galie PA, Chen CS. Biomimetic model to reconstitute angio-
genic sprouting morphogenesis in vitro. Proc Natl Acad Sci USA. 2013;110:6712–7.
35. Song JW, Daubriac J, Tse JM, Bazou D, Munn LL. RhoA mediates flow-induced endothelial sprouting in a 3-D tissue
analogue of angiogenesis. Lab Chip. 2012;12:5000–6.
36. Sato T, Clevers H. Growing self-organizing mini-guts from a single intestinal stem cell: mechanism and applica-
tions. Science. 2013;340:1190–4.
37. Sellgren KL, Hawkins BT, Grego S. An optically transparent membrane supports shear stress studies in a three-
dimensional microfluidic neurovascular unit model. Biomicrofluidics. 2015;9:687.
38. Yang SH, Jin WC, Huh D, Jo HA, Kim S, Lim CS, Lee JC, Kim HC, Kwon HM, Chang WJ. Roles of fluid shear stress and
retinoic acid in the differentiation of primary cultured human podocytes. Exp Cell Res. 2017;354:48–56.
39. Kshitiz, Park J, Kim P, Helen W, Engler AJ, Levchenko A, Kim DH. Control of stem cell fate and function by engineer-
ing physical microenvironments. Integr Biol. 2012;4:1008.
40. Jang KJ, Cho HS, Kang DH, Bae WG, Kwon TH, Suh KY. Fluid-shear-stress-induced translocation of aquaporin-2 and
reorganization of actin cytoskeleton in renal tubular epithelial cells. Integr Biol. 2010;3:134–41.
41. Zhou J, Khodakov DA, Ellis AV, Voelcker NH. Surface modification for PDMS-based microfluidic devices. Electropho-
resis. 2012;33:89–104.
42. Tibbe MP, Leferink AM, van den Berg A, Eijkel JCT, Segerink LI. Microfluidic gel patterning method by use of a tem-
porary membrane for organ-on-chip applications. Adv Mater Technol. 2018;3:1700200. https​://[Link]/10.1002/
admt.20170​0200.
43. Xue D, Wang Y, Zhang J, Mei D, Wang Y, Chen S. Projection-based 3D printing of cell patterning Scaffolds with
multiscale channels. ACS Appl Mater Interfaces. 2018;10:19428–35. https​://[Link]/10.1021/acsam​i.8b038​67.
44. Li Y-C, Lin M-W, Yen M-H, Fan SM-Y, Wu J-T, Young T-H, Cheng J-Y, Lin S-J. Programmable laser-assisted surface
microfabrication on a poly(vinyl alcohol)-coated glass chip with self-changing cell adhesivity for heterotypic cell
patterning. ACS Appl Mater Interfaces. 2015;7:22322–32. https​://[Link]/10.1021/acsam​i.5b059​78.
45. Mandenius C-F. Conceptual design of micro-bioreactors and organ-on-chips for studies of cell cultures. Bioengi-
neering (Basel). 2018. https​://[Link]/10.3390/bioen​ginee​ring5​03005​6.
46. Sun X, Nunes SS. Maturation of human stem cell-derived cardiomyocytes in biowires using electrical stimulation. J
Vis Exp Jove. 2017;2017(123). https​://[Link]/10.3791/55373​.
47. Yang PC, Qi Y, Zhang DH. Studies, bottlenecks and challenges of microarray of micro organs. Chin J Tissue Eng Res.
2018;22:5234–40. https​://[Link]/10.3969/[Link].2095-4344.0558.
48. Peel S, Corrigan AM, Ehrhardt B, Jang K-J, Caetano-Pinto P, Boeckeler M, Rubins JE, Kodella K, Petropolis DB, Ronxhi
J, et al. Introducing an automated high content confocal imaging approach for organs-on-chips. Lab Chip.
2019;19:410–21. https​://[Link]/10.1039/c8lc0​0829a​.
49. Kane KIW, Moreno EL, Hachi S, Walter M, Jarazo J, Oliveira MAP, Hankemeier T, Vulto P, Schwamborn JC, Thoma M,
et al. Automated microfluidic cell culture of stem cell derived dopaminergic neurons. Sci Rep. 2019;9:1796. https​://
[Link]/10.1038/s4159​8-018-34828​-3.
50. Mccuskey RS. The hepatic microvascular system in health and its response to toxicants. Anat Rec.
2010;291:661–71.
51. Cho CH, Park J, Tilles AW, Berthiaume F, Toner M, Yarmush ML. Layered patterning of hepatocytes in co-culture
systems using microfabricated stencils. Biotechniques. 2018;48:47–52.
52. Kane BJ, Zinner MJ, Yarmush ML, Toner M. Liver-specific functional studies in a microfluidic array of primary mam-
malian hepatocytes. Anal Chem. 2006;78:4291–8. https​://[Link]/10.1021/ac051​856v.
53. Lee PJ, Hung PJ, Lee LP. An artificial liver sinusoid with a microfluidic endothelial-like barrier for primary hepatocyte
culture. Biotechnol Bioeng. 2007;97:1340–6. https​://[Link]/10.1002/bit.21360​.
54. Hegde M, Jindal R, Bhushan A, Bale SS, Mccarty WJ, Golberg I, Usta OB, Yarmush ML. Dynamic interplay of flow and
collagen stabilizes primary hepatocytes culture in a microfluidic platform. Lab Chip. 2014;14:2033–9.
55. Fan A, Qu Y, Liu X, Zhong R, Yong L. Organ-on-a-chip: new platform for biological analysis. Anal Chem Insights.
2015;10:39–45.
56. Ma L-D, Wang Y-T, Wang J-R, Wu J-L, Meng X-S, Hu P, Mu X, Liang Q-L, Luo G-A. Design and fabrication of a liver-
on-a-chip platform for convenient, highly efficient, and safe in situ perfusion culture of 3D hepatic spheroids. Lab
Chip. 2018;18:2547–62. https​://[Link]/10.1039/c8lc0​0333e​.
57. Yum K, Hong SG, Healy KE, Lee LP. Physiologically relevant organs on chips (pages 16–27). Biotechnol J.
2014;9:16–27.
58. Riahi R, Shaegh SAM, Ghaderi M, Zhang YS, Su RS, Aleman J, Massa S, Kim D, Dokmeci MR, Khademhosseini A.
Automated microfluidic platform of bead-based electrochemical immunosensor integrated with bioreactor for
continual monitoring of cell secreted biomarkers. Sci Rep. 2016;6:24598.
 Wu et al. BioMed Eng OnLine (2020) 19:9 Page 17 of 19

59. Chong LH, Li H, Wetzel I, Cho H, Toh Y-C. A liver-immune coculture array for predicting systemic drug-induced
skin sensitization. Lab Chip. 2018;18:3239–50. https​://[Link]/10.1039/c8lc0​0790j​.
60. Lu S, Cuzzucoli F, Jiang J, Liang L-G, Wang Y, Kong M, Zhao X, Cui W, Li J, Wang S. Development of a biomimetic
liver tumor-on-a-chip model based on decellularized liver matrix for toxicity testing. Lab Chip. 2018;18:3379–
92. https​://[Link]/10.1039/c8lc0​0852c​.
61. Kang YBA, Sodunke TR, Lamontagne J, Cirillo J, Rajiv C, Bouchard MJ, Noh M. Liver sinusoid on a chip: long-
term layered co-culture of primary rat hepatocytes and endothelial cells in microfluidic platforms. Biotechnol
Bioeng. 2015;112:2571–82.
62. Zhou Q, Patel D, Kwa T, Haque A, Matharu Z, Stybayeva G, Gao Y, Diehl AM, Revzin A. Liver injury-on-a-chip:
microfluidic co-cultures with integrated biosensors for monitoring liver cell signaling during injury. Lab Chip.
2015;15:4467–78. https​://[Link]/10.1039/C5LC0​0874C​.
63. Guenat OT, Berthiaume F. Incorporating mechanical strain in organs-on-a-chip: lung and skin. Biomicrofluidics.
2018;12:42207. https​://[Link]/10.1063/1.50248​95.
64. Huh D, Matthews BD, Mammoto A, Montoya-Zavala M, Hsin HY, Ingber DE. Reconstituting organ-level lung
functions on a chip. Science. 2010;328:1662–8. https​://[Link]/10.1126/scien​ce.11883​02.
65. Dongeun H, Leslie DC, Matthews BD, Fraser JP, Samuel J, Hamilton GA, Thorneloe KS, Michael Allen M, Ingber
DE. A human disease model of drug toxicity-induced pulmonary edema in a lung-on-a-chip microdevice. Sci
Transl Med. 2012;4:159ra147.
66. Stucki AO, Stucki JD, Hall SRR, Felder M, Mermoud Y, Schmid RA, Geiser T, Guenat OT. A lung-on-a-chip array
with an integrated bio-inspired respiration mechanism. Lab Chip. 2015;15:1302–10. https​://[Link]/10.1039/
c4lc0​1252f​.
67. Blume C, Reale R, Held M, Millar TM, Collins JE, Davies DE, Morgan H, Swindle EJ. Temporal monitoring of
differentiated human airway epithelial cells using microfluidics. PLoS ONE. 2015;10:e0139872. https​://doi.
org/10.1371/journ​[Link].01398​72.
68. Humayun M, Chow C-W, Young EWK. Microfluidic lung airway-on-a-chip with arrayable suspended gels for
studying epithelial and smooth muscle cell interactions. Lab Chip. 2018;18:1298–309. https​://[Link]/10.1039/
c7lc0​1357d​.
69. Yang X, Li K, Zhang X, Liu C, Guo B, Wen W, Gao X. Nanofiber membrane supported lung-on-a-chip microde-
vice for anti-cancer drug testing. Lab Chip. 2018;18:486–95. https​://[Link]/10.1039/c7lc0​1224a​.
70. Peng J, Rochow N, Dabaghi M, Bozanovic R, Jansen J, Predescu D, DeFrance B, Lee SY, Fusch G, Ravi Selvagana-
pathy P, et al. Postnatal dilatation of umbilical cord vessels and its impact on wall integrity: prerequisite for the
artificial placenta. Int J Artif Organs. 2018;41:393–9.
71. Dabaghi M, Fusch G, Saraei N, Rochow N, Brash JL, Fusch C, Ravi Selvaganapathy P. An artificial placenta type
microfluidic blood oxygenator with double-sided gas transfer microchannels and its integration as a neonatal
lung assist device. Biomicrofluidics. 2018;12:44101. https​://[Link]/10.1063/1.50347​91.
72. Xu Z, Gao Y, Hao Y, Li E, Wang Y, Zhang J, Wang W, Gao Z, Wang Q. Application of a microfluidic chip-based 3D
co-culture to test drug sensitivity for individualized treatment of lung cancer. Biomaterials. 2013;34:4109–17.
73. Benam KH, Villenave R, Lucchesi C, Varone A, Ingber DE. Small airway-on-a-chip enables analysis of human
lung inflammation and drug responses in vitro. Nat Methods. 2015;13:151.
74. Jang K-J, Suh K-Y. A multi-layer microfluidic device for efficient culture and analysis of renal tubular cells. Lab
Chip. 2010;10:36–42. https​://[Link]/10.1039/b9075​15a.
75. Jang K-J, Mehr AP, Hamilton GA, Mcpartlin LA, Chung S, Suh K-Y, Ingber DE. Human kidney proximal tubule-
on-a-chip for drug transport and nephrotoxicity assessment. Integr Biol (Camb). 2013;5:1119–29. https​://doi.
org/10.1039/c3ib4​0049b​.
76. Musah S, Dimitrakakis N, Camacho DM, Church GM, Ingber DE. Directed differentiation of human induced
pluripotent stem cells into mature kidney podocytes and establishment of a glomerulus chip. Nat Protoc.
2018;13:1662–85. https​://[Link]/10.1038/s4159​6-018-0007-8.
77. Sakolish CM, Philip B, Mahler GJ. A human proximal tubule-on-a-chip to study renal disease and toxicity.
Biomicrofluidics. 2019;13:14107. https​://[Link]/10.1063/1.50831​38.
78. Schutgens F, Rookmaaker MB, Margaritis T, Rios A, Ammerlaan C, Jansen J, Gijzen L, Vormann M, Vonk A,
Viveen M, et al. Tubuloids derived from human adult kidney and urine for personalized disease modeling. Nat
Biotechnol. 2019;37:303–13. https​://[Link]/10.1038/s4158​7-019-0048-8.
79. Nieskens TTG, Sjögren A-K. Emerging in vitro systems to screen and predict drug-induced kidney toxicity.
Semin Nephrol. 2019;39:215–26. https​://[Link]/10.1016/[Link]​phrol​.2018.12.009.
80. Visone R, Gilardi M, Marsano A, Rasponi M, Bersini S, Moretti M. Cardiac meets skeletal: what’s new in microflu-
idic models for muscle tissue engineering. Molecules. 2016. https​://[Link]/10.3390/molec​ules2​10911​28.
81. Grosberg A, Nesmith AP, Goss JA, Brigham MD, McCain ML, Parker KK. Muscle on a chip: in vitro contractility assays
for smooth and striated muscle. J Pharmacol Toxicol Methods. 2012;65:126–35. https​://[Link]/10.1016/[Link]​
.2012.04.001.
82. Zhang D, Shadrin I, Lam J, Xian HQ, Snodgrass R, Bursac N. Tissue-engineered cardiac patch for advanced func-
tional maturation of human ESC-derived cardiomyocytes. Biomaterials. 2013;34:5813–20.
83. Zhang YS, Arneri A, Bersini S, Shin S-R, Zhu K, Goli-Malekabadi Z, Aleman J, Colosi C, Busignani F, Dell’Erba V, et al.
Bioprinting 3D microfibrous scaffolds for engineering endothelialized myocardium and heart-on-a-chip. Biomate-
rials. 2016;110:45–59. https​://[Link]/10.1016/[Link]​teria​ls.2016.09.003.
84. Zhang X, Wang T, Wang P, Hu N. High-throughput assessment of drug cardiac safety using a high-speed imped-
ance detection technology-based heart-on-a-chip. Micromachines (Basel). 2016. https​://[Link]/10.3390/mi707​
0122.
85. Marsano A, Conficconi C, Lemme M, Occhetta P, Gaudiello E, Votta E, Cerino G, Redaelli A, Rasponi M. Beating heart
on a chip: a novel microfluidic platform to generate functional 3D cardiac microtissues. Lab Chip. 2016;16:599–
610. https​://[Link]/10.1039/c5lc0​1356a​.
Wu et al. BioMed Eng OnLine (2020) 19:9 Page 18 of 19

86. Schneider O, Zeifang L, Fuchs S, Sailer C, Loskill P. User-friendly and parallelized generation of human induced
pluripotent stem cell-derived microtissues in a centrifugal heart-on-a-chip. Tissue Eng Part A. 2019;25:786–98.
https​://[Link]/10.1089/[Link].2019.0002.
87. Tzatzalos E, Abilez OJ, Shukla P, Wu JC. Engineered heart tissues and induced pluripotent stem cells: macro-
and microstructures for disease modeling, drug screening, and translational studies. Adv Drug Deliv Rev.
2016;96:234–44.
88. Kang TH, Kim HJ. Farewell to animal testing: innovations on human intestinal microphysiological systems.
Micromachines (Basel). 2016. https​://[Link]/10.3390/mi707​0107.
89. Imura Y, Asano Y, Sato K, Yoshimura E. A microfluidic system to evaluate intestinal absorption. Anal Sci.
2009;2009(25):1403–7.
90. Sung JH, Yu J, Luo D, Shuler ML, March JC. Microscale 3-D hydrogel scaffold for biomimetic gastrointestinal (GI)
tract model. Lab Chip. 2011;11:389–92. https​://[Link]/10.1039/c0lc0​0273a​.
91. Kim HJ, Huh D, Hamilton G, Ingber DE. Human gut-on-a-chip inhabited by microbial flora that experiences intesti-
nal peristalsis-like motions and flow. Lab Chip. 2012;12:2165–74. https​://[Link]/10.1039/c2lc4​0074j​.
92. Kim HJ, Li H, Collins JJ, Ingber DE. Contributions of microbiome and mechanical deformation to intestinal bacterial
overgrowth and inflammation in a human gut-on-a-chip. Proc Natl Acad Sci USA. 2016;113:E7–15. https​://doi.
org/10.1073/pnas.15221​93112​.
93. Kasendra M, Tovaglieri A, Sontheimer-Phelps A, Jalili-Firoozinezhad S, Bein A, Chalkiadaki A, Scholl W, Zhang C,
Rickner H, Richmond CA, et al. Development of a primary human small intestine-on-a-chip using biopsy-derived
organoids. Sci Rep. 2018;8:2871. https​://[Link]/10.1038/s4159​8-018-21201​-7.
94. Vandussen KL, Marinshaw JM, Nurmohammad S, Hiroyuki M, Clara M, Tarr PI, Ciorba MA, Stappenbeck TS. Devel-
opment of an enhanced human gastrointestinal epithelial culture system to facilitate patient-based assays. Gut.
2014;64:911.
95. Jalili-Firoozinezhad S, Gazzaniga FS, Calamari EL, Camacho DM, Fadel CW, Bein A, Swenor B, Nestor B, Cronce MJ,
Tovaglieri A, et al. A complex human gut microbiome cultured in an anaerobic intestine-on-a-chip. Nat Biomed
Eng. 2019. https​://[Link]/10.1038/s4155​1-019-0397-0.
96. Shin W, Hinojosa CD, Ingber DE, Kim HJ. Human intestinal morphogenesis controlled by transepithelial morpho-
gen gradient and flow-dependent physical cues in a microengineered gut-on-a-chip. iScience. 2019;15:391–406.
97. Lee SH, Sung JH. Microtechnology-based multi-organ models. Bioengineering (Basel). 2017. https​://doi.
org/10.3390/bioen​ginee​ring4​02004​6.
98. Marx U, Walles H, Hoffmann S, Lindner G, Horland R, Sonntag F, Klotzbach U, Sakharov D, Tonevitsky A, Lauster
R. ‘Human-on-a-chip’ developments: a translational cutting-edge alternative to systemic safety assessment and
efficiency evaluation of substances in laboratory animals and man? Altern Lab Anim. 2012;40:235–57. https​://doi.
org/10.1177/02611​92912​04000​504.
99. Zhang B, Montgomery M, Chamberlain MD, Ogawa S, Korolj A, Pahnke A, Wells LA, Massé S, Kim J, Reis L, et al.
Biodegradable scaffold with built-in vasculature for organ-on-a-chip engineering and direct surgical anastomosis.
Nat Mater. 2016;15:669–78. https​://[Link]/10.1038/nmat4​570.
100. Palaninathan V, Kumar V, Maekawa T, Liepmann D, Paulmurugan R, Eswara JR, Ajayan PM, Augustine S, Malhotra
BD, Viswanathan S, et al. Multi-organ on a chip for personalized precision medicine. MRC. 2018;8:652–67. https​://
[Link]/10.1557/mrc.2018.148.
101. Zhao Y, Kankala RK, Wang S-B, Chen A-Z. Multi-organs-on-chips: towards long-term biomedical investigations.
Molecules. 2019. https​://[Link]/10.3390/molec​ules2​40406​75.
102. Rogal J, Probst C, Loskill P. Integration concepts for multi-organ chips: how to maintain flexibility?! Future Sci OA.
2017;3:FSO180. https​://[Link]/10.4155/fsoa-2016-0092.
103. Wagner I, Materne E-M, Brincker S, Süssbier U, Frädrich C, Busek M, Sonntag F, Sakharov DA, Trushkin EV, Tonevitsky
AG, et al. A dynamic multi-organ-chip for long-term cultivation and substance testing proven by 3D human liver
and skin tissue co-culture. Lab Chip. 2013;13:3538–47. https​://[Link]/10.1039/c3lc5​0234a​.
104. van Midwoud PM, Merema MT, Verpoorte E, Groothuis GMM. A microfluidic approach for in vitro assessment of
interorgan interactions in drug metabolism using intestinal and liver slices. Lab Chip. 2010;10:2778–86. https​://doi.
org/10.1039/c0lc0​0043d​.
105. Tsamandouras N, Wen LKC, Edington CD, Stokes CL, Griffith LG, Cirit M. Integrated gut and liver microphysiological
systems for quantitative in vitro pharmacokinetic studies. Aaps J. 2017;19:1–14.
106. Skardal A, Murphy SV, Devarasetty M, Mead I, Kang H-W, Seol Y-J, Shrike Zhang Y, Shin S-R, Zhao L, Aleman J, et al.
Multi-tissue interactions in an integrated three-tissue organ-on-a-chip platform. Sci Rep. 2017;7:8837. https​://doi.
org/10.1038/s4159​8-017-08879​-x.
107. Maschmeyer I, Hasenberg T, Jaenicke A, Lindner M, Lorenz AK, Zech J, Garbe L-A, Sonntag F, Hayden P, Ayehunie
S, et al. Chip-based human liver-intestine and liver-skin co-cultures–A first step toward systemic repeated dose
substance testing in vitro. Eur J Pharm Biopharm. 2015;95:77–87. https​://[Link]/10.1016/[Link].2015.03.002.
108. Maschmeyer I, Lorenz AK, Schimek K, Hasenberg T, Ramme AP, Hübner J, Lindner M, Drewell C, Bauer S, Thomas A,
et al. A four-organ-chip for interconnected long-term co-culture of human intestine, liver, skin and kidney equiva-
lents. Lab Chip. 2015;15:2688–99. https​://[Link]/10.1039/c5lc0​0392j​.
109. Oleaga C, Bernabini C, Smith AST, Srinivasan B, Jackson M, McLamb W, Platt V, Bridges R, Cai Y, Santhanam N, et al.
Multi-Organ toxicity demonstration in a functional human in vitro system composed of four organs. Sci Rep.
2016;6:20030. https​://[Link]/10.1038/srep2​0030.
110. Edington CD, Chen WLK, Geishecker E, Kassis T, Soenksen LR, Bhushan BM, Freake D, Kirschner J, Maass C, Tsaman-
douras N, et al. Interconnected microphysiological systems for quantitative biology and pharmacology studies. Sci
Rep. 2018;8:4530. https​://[Link]/10.1038/s4159​8-018-22749​-0.
111. Lee H, Kim DS, Ha SK, Choi I, Lee JM, Sung JH. A pumpless multi-organ-on-a-chip (MOC) combined with a phar-
macokinetic-pharmacodynamic (PK-PD) model. Biotechnol Bioeng. 2017;114:432–43. https​://[Link]/10.1002/
bit.26087​.
 Wu et al. BioMed Eng OnLine (2020) 19:9 Page 19 of 19

112. Satoh T, Sugiura S, Shin K, Onuki-Nagasaki R, Ishida S, Kikuchi K, Kakiki M, Kanamori T. A multi-throughput multi-
organ-on-a-chip system on a plate formatted pneumatic pressure-driven medium circulation platform. Lab Chip.
2017;18:115–25. https​://[Link]/10.1039/c7lc0​0952f​.
113. Díaz Lantada A, Pfleging W, Besser H, Guttmann M, Wissmann M, Plewa K, Smyrek P, Piotter V, García-Ruíz JP.
Research on the methods for the mass production of multi-scale organs-on-chips. Polymers (Basel). 2018. https​://
[Link]/10.3390/polym​10111​238.
114. Wang Y, Huang X, Shen Y, Hang R, Zhang X, Wang Y, Yao X, Tang B. Direct writing alginate bioink inside pre-poly-
mers of hydrogels to create patterned vascular networks. J Mater Sci. 2019;54:7883–92. https​://[Link]/10.1007/
s1085​3-019-03447​-2.
115. Hong S, Kang EY, Byeon J, Jung S-H, Hwang C. Embossed membranes with vascular patterns guide vascularization
in a 3D tissue model. Polymers (Basel). 2019. https​://[Link]/10.3390/polym​11050​792.
116. Torras N, García-Díaz M, Fernández-Majada V, Martínez E. Mimicking epithelial tissues in three-dimensional cell
culture models. Front Bioeng Biotechnol. 2018;6:197. https​://[Link]/10.3389/fbioe​.2018.00197​.
117. Alexander FA, Eggert S, Wiest J. Skin-on-a-chip: transepithelial electrical resistance and extracellular acidification
measurements through an automated air-liquid interface. Genes (Basel). 2018. https​://[Link]/10.3390/genes​
90201​14.
118. Wang YI, Abaci HE, Shuler ML. Microfluidic blood-brain barrier model provides in vivo-like barrier properties for
drug permeability screening. Biotechnol Bioeng. 2017;114:184–94. https​://[Link]/10.1002/bit.26045​.
119. Wevers NR, Kasi DG, Gray T, Wilschut KJ, Smith B, van Vught R, Shimizu F, Sano Y, Kanda T, Marsh G, et al. A perfused
human blood-brain barrier on-a-chip for high-throughput assessment of barrier function and antibody transport.
Fluids Barriers CNS. 2018;15:23. https​://[Link]/10.1186/s1298​7-018-0108-3.
120. Khodabukus A, Madden L, Prabhu NK, Koves TR, Jackman CP, Muoio DM, Bursac N. Electrical stimulation increases
hypertrophy and metabolic flux in tissue-engineered human skeletal muscle. Biomaterials. 2019;198:259–69. https​
://[Link]/10.1016/[Link]​teria​ls.2018.08.058.
121. Kim W, Kim J, Park H-S, Jeon JS. Development of microfluidic stretch system for studying recovery of damaged
skeletal muscle cells. Micromachines (Basel). 2018. https​://[Link]/10.3390/mi912​0671.
122. Yildirimer L, Zhang Q, Kuang S, Cheung C-WJ, Chu KA, He Y, Yang M, Zhao X. Engineering three-dimensional
microenvironments towards in vitro disease models of the central nervous system. Biofabrication. 2019. https​://
[Link]/10.1088/1758-5090/ab17a​a.
123. Choi J-H, Cho H-Y, Choi J-W. Microdevice platform for in vitro nervous system and its disease model. Bioengineer-
ing (Basel). 2017. https​://[Link]/10.3390/bioen​ginee​ring4​03007​7.
124. Zhang J, Wei X, Zeng R, Xu F, Li X. Stem cell culture and differentiation in microfluidic devices toward organ-on-a-
chip. Future Sci OA. 2017;3:FSO187. https​://[Link]/10.4155/fsoa-2016-0091.
125. Wnorowski A, Yang H, Wu JC. Progress, obstacles, and limitations in the use of stem cells in organ-on-a-chip mod-
els. Adv Drug Deliv Rev. 2019;140:3–11. https​://[Link]/10.1016/[Link].2018.06.001.
126. Pittenger MF. Multilineage potential of adult human mesenchymal stem cells. Science. 1999;284:143–7.
127. Wagner W, Ho AD. Mesenchymal stem cell preparations—comparing apples and oranges. Stem Cell Reviews.
2007;3:239–48. https​://[Link]/10.1007/s1201​5-007-9001-1.
128. Thomson JA, Itskovitzeldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM. Embryonic stem cell lines
derived from human blastocysts. Science. 1998;282:1145.
129. Becker H, Hansen-Hagge T, Kurtz A, Mrowka R, Wölfl S, Gärtner C. Microfluidic devices for stem-cell cultivation, dif-
ferentiation and toxicity testing. In: Gray BL, Becker H, editors. Microfluidics, BioMEMS, and medical microsystems
XV. San Francisco: SPIE BiOS; 2017. p. 1006116.
130. Scott CW, Peters MF, Dragan YP. Human induced pluripotent stem cells and their use in drug discovery for toxicity
testing. Toxicol Lett. 2013;219:49–58.
131. Narsinh KH, Jordan P, Wu JC. Comparison of human induced pluripotent and embryonic stem cells: fraternal or
identical twins? Mol Ther. 2011;19:635.
132. Zhao M-T, Chen H, Liu Q, Shao N-Y, Sayed N, Wo H-T, Zhang JZ, Ong S-G, Liu C, Kim Y, et al. Molecular and func-
tional resemblance of differentiated cells derived from isogenic human iPSCs and SCNT-derived ESCs. Proc Natl
Acad Sci. 2017;114:E11111–20. https​://[Link]/10.1073/pnas.17089​91114​.
133. Park D, Lim J, Park JY, Lee S-H. Concise review: stem cell microenvironment on a chip: current technologies
for tissue engineering and stem cell biology. Stem Cells Transl Med. 2015;4:1352–68. https​://[Link]/10.5966/
sctm.2015-0095.
134. Qian T, Shusta EV, Palecek SP. Advances in microfluidic platforms for analyzing and regulating human pluripotent
stem cells. Curr Opin Genet Dev. 2015;34:54–60. https​://[Link]/10.1016/[Link].2015.07.007.

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