PROGRESS REPORT
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Is it Time for Reviewer 3 to Request Human Organ Chip
Experiments Instead of Animal Validation Studies?
Donald E. Ingber
For the past century, experimental data obtained from animal studies have
been required by reviewers of scientific articles and grant applications to
validate the physiological relevance of in vitro results. At the same time,
pharmaceutical researchers and regulatory agencies recognize that results
from preclinical animal models frequently fail to predict drug responses in
humans. This Progress Report reviews recent advances in human
organ-on-a-chip (Organ Chip) microfluidic culture technology, both with
single Organ Chips and fluidically coupled human “Body-on-Chips”
platforms, which demonstrate their ability to recapitulate human physiology
and disease states, as well as human patient responses to clinically relevant
drug pharmacokinetic exposures, with higher fidelity than other in vitro
models or animal studies. These findings raise the question of whether
continuing to require results of animal testing for publication or grant funding
still makes scientific or ethical sense, and if more physiologically relevant
human Organ Chip models might better serve this purpose. This issue is
addressed in this article in context of the history of the field, and advantages
and disadvantages of Organ Chip approaches versus animal models are
discussed that should be considered by the wider research community.
1. Introduction
Biomedical research grant applications and manuscripts com-
monly begin with an explanation of the potential clinical rel-
evance of the work for human health. Most basic researchers
carry out experiments using in vitro models, and thus, key to
D. E. Ingber
Wyss Institute for Biologically Inspired Engineering at Harvard University
Boston, MA 02115, USA
E-mail: don.ingber@wyss.harvard.edu
D. E. Ingber
Vascular Biology Program, Department of Surgery
Boston Children’s Hospital and Harvard Medical School
Boston, MA 02115, USA
D. E. Ingber
Harvard John A. Paulson School of Engineering and Applied Sciences
Cambridge, MA 02138, USA
The ORCID identification number(s) for the author(s) of this article
can be found under https://doi.org/10.1002/advs.202002030
© 2020 The Authors. Published by Wiley-VCH GmbH. This is an open
access article under the terms of the Creative Commons Attribution
License, which permits use, distribution and reproduction in any
medium, provided the original work is properly cited.
DOI: 10.1002/advs.202002030
Adv. Sci. 2020, 2002030 2002030 (1 of 15)
their premise is that the results they gen-
erate in their cell cultures will translate to
humans. Because in vitro studies generally
lack the natural three dimensional (3D) con-
text, vascular flow, and physico-chemical
microenvironment of living tissues and or-
gans, as well as the multi-organ physiol-
ogy of whole organisms, many question the
clinical relevance of findings obtained with
these simplified models. For this reason,
most researchers who submit a grant ap-
plication or publication based on in vitro
findings commonly expect to find at least
one reviewer (the classic exasperating “Re-
viewer 3”) who demands that additional an-
imal experiments be carried out to validate
their findings before the work could be ac-
ceptable for publication or funding. This ar-
ticle seeks to provoke a conversation in the
scientific community by asking two simple
questions: does this make sense, and if not,
is there a better alternative? I address these
questions by reviewing recent progress that
has been made using organoids and engi-
neered microphysiological systems (MPS)
with a focus on microfluidic organ-on-a-
chip (Organ Chip) culture technologies.
2. Why Do We Require Animal Studies to Validate
In Vitro Findings?
Animal experiments have been the mainstay of scientific re-
search since the time of Aristotle, and there are good reasons
for this. Many molecular mechanisms, physiological processes,
and disease states involve complex tissue- and organ-level struc-
tures and functions, as well as interplay among multiple organ
systems. The effects that drugs produce in our bodies not only
depend on their effects within individual cells; they are also gov-
erned by their pharmacokinetics (PK), which defines how their
levels change over time in flowing blood and in different tis-
sues depending on the dynamics of their absorption, distribu-
tion, metabolism, and excretion (ADME) behaviors. It is not pos-
sible to model these complex responses, disease phenotypes, and
PK behaviors in vitro using conventional culture technology, and
hence, it has been difficult to study the relation between molec-
ular mechanism, pathogenesis, and effects of drugs or toxins in
a meaningful way without carrying out animal experiments. An-
other excellent reason for doing animal studies is when experi-
ments in humans would be unethical, for example, when testing
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the effects of exposure to lethal toxins, high energy radiation, or
potentially deadly pathogens, such as infection by Ebola or the
SARS-CoV-2 virus.
Reviewers of grants and publications appreciate the impor-
tance of animal models for all of the above reasons, but per-
haps more central to their argument is the ability of animals, and
particularly mice, to be genetically engineered to mimic human
disease states. This has been one of the central tenets of basic
biomedical research for the past 40 years, particularly in studies
focused on discovery of new therapeutics to alleviate human suf-
fering. But are animal models good predictors of human patient
outcomes? The answer to this question might be surprising.
Studies by the pharmaceutical industry, as well as regula-
tory and government agencies around the world, including the
United States Food and Drug Administration (FDA) and National
Institutes of Health (NIH), have consistently found that results
from drug testing in preclinical animal models are poor predic-
tors of human responses.[1–3] Estimates of failure rates range as
high 90%, and this relates to both lack of efficacy and safety prob-
lems when the drugs are evaluated in human clinical trials.
Many animal models, including genetically engineered mice,
can mimic human disease phenotypes; however, the underlying
molecular, cellular, and physiological mechanisms are often dis-
tinct. For example, while mice can generate phenotypes reminis-
cent of multi-system inflammatory responses in humans, such as
sepsis and acute respiratory distress (ARDS), the genomic path-
ways that underlie these responses differ significantly.[4] Simi-
larly, while mice can be engineered to develop neurofibrillary
tangles similar to those seen in the brains of humans with
Alzheimer’s Disease, the molecular mechanisms uncovered in
mouse models and drugs developed based on these insights have
not translated well when the drugs were moved into the clinic.[5]
Perhaps this is why hundreds of drugs that were found to be ef-
fective when tested on animals for diseases ranging from stroke
to inflammation were not found to be active in humans, and why
many vaccines have similarly failed in clinical trials even after
working well in non-human primates.[6] Furthermore, recent ad-
vances in the biotechnology industry have led to biologic thera-
pies, such as therapeutic monoclonal antibodies, that are so spe-
cific for their human targets that they do not even cross react with
related molecules in non-human primates, and so there is no
other option than to test them in a human model. Finally, a major
issue that has always concerned the field of drug development is
that there may be many therapies out there that were killed be-
cause they failed to produce positive results in animals, yet they
could have potentially saved human lives. So why is Reviewer 3
still demanding that animal studies be carried out before scien-
tific findings can be shared openly with the wider community or
funding is provided to advance a potentially important research
program? It is likely because they feel that there is no alternative;
but this is not the case.
3. Are there Viable In Vitro Alternatives to Animal
Models?
3.1. Human Organoids
Over the past decade, there have been many advances in culture
technology that provide levels of cytodifferentiation, tissue func-
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tionalities, and organ-level responses not possible in the past.
Organoids, for example, are clusters of cells that contain a sub-
set of self-renewing stem cells, which grow and self-organize into
small closed spheres that undergo cytodifferentiation and recre-
ate 3D tissue-like structures and functions seen in living organs
when grown embedded within solid extracellular matrix (ECM)
gels in vitro (Figure 1).[7] Methods for generating organoids have
been published for many organs, including intestine, lung, liver,
kidney, and brain, among others, and because they contain stem
cells, they can be used to continuously expand organ-specific hu-
man cell populations for additional in vitro studies.
Importantly, organoids generated with human patient-derived
cells or human-induced pluripotent stem (iPS) cells have proven
to be extremely valuable for studying mechanisms relating to dif-
ferentiation, morphogenesis, pathogenesis, and drug action, as
well as discovery of disease biomarkers that act at the cell and tis-
sue levels.[7] Thus, they have a key role to play in the preclinical
drug development pipeline, and they may be used to replace an-
imal studies that are focused on cell and molecular mechanisms
of disease.
However, because organoids are closed structures that lack
tissue–tissue interfaces, vascular flow, circulating immune cells,
and physiologically relevant mechanical cues, they cannot fully
recapitulate organ-level responses or be used to study drug ef-
fects under pharmacologically relevant conditions that depend
upon dynamic drug PK exposure profiles. Integration of self-
organized capillary networks within 3D organoid cultures[8] and
addition of immune cells to the surrounding ECM gel[9] may help
to overcome some of these limitations. But there are still signif-
icant limitations, such as an inability control vascular architec-
ture and flow dynamics, and hence how chemicals, inflamma-
tory molecules, drugs, and immune cells are transported to and
from these tissues as they are within living vascularized organs in
our bodies. Because organoids are closed structures surrounded
by a thick dense ECM, it is also difficult to measure of transport
and absorption of nutrients, chemicals, or drugs across the ep-
ithelial tissue, experimentally sample contents of the internal lu-
men, sustain co-cultures with living microbiome, and integrate
sensors for on-line functional measurement using these models.
3.2. Static Microphysiological Systems
Over the past decade, there also has been an explosion in the de-
velopment of advanced MPS, such as engineered 3D tissues and
microfluidic Organ Chips with dynamic flow. Some static MPS
models that lack fluid flow can produce high levels of function-
ality and mimicry of tissue functions, and they have been used
in combination with primary human cells or iPS-derived cells
from patients to gain insight into human physiology and patho-
genesis that would be difficult to accomplish using animal mod-
els. For example, contractile heart tissues engineered using iPS
cell-derived cardiomyocytes from patients with Barth syndrome
were able to mimic key features of the cardiomyopathy these pa-
tients experience in vitro, and this work led to new mechanis-
tic insights into the pathogenesis of this disease.[10] Tissue engi-
neering with iPS-derived cardiomyocytes from human patients
along with physiological electrical conditioning also enabled de-
velopment of electrophysiologically distinct atrial and ventricular
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Figure 1. Potential in vitro replacements for animal testing. Schematics of mice, organoids that grow as small closed spheres which undergo organ-
specific cyto- and histo-differentiation within 3D ECM gels, and microfluidic Organ Chips that may be lined with cells from organoids (dashed line),
iPS cell-derived cells, or primary cells. Organ Chips reconstitute tissue–tissue interfaces between organ-specific epithelium and endothelium, vascular
perfusion, interstitial flow or air–liquid interfaces, and mechanical cues (e.g., by applying cyclic strain via application of suction to hollow side chambers)
to mimic breathing or peristalsis motions. Circulating or resident immune cells, connective tissue cells, nerve cells, and other cell types can be integrated
into the Organ Chips as needed to recapitulate increasing levels of complexity.

tissues that mimicked chamber-specific drug responses and gene
expression, which permitted modeling of polygenic left ventricu-
lar hypertrophy in vitro.[11] There are also numerous other exam-
ples of how static MPS models can mimic human cell and tissue
pathophysiology, as well as cellular responses to drug therapies,
and so they too represent viable replacements for some animal
studies.[12] However, these MPS models do not enable dynamic
flow required to mimic drug PK or support multi-organ coupling,
and hence, they only satisfy a subset of requirements for replac-
ing animal studies, much like organoids.
3.3. Microfluidic Organ Chips
Microfluidic Organ Chips are a form of dynamic MPS that reca-
pitulate vascular perfusion, tissue–tissue interfaces, and organ-
relevant mechanical motions, while also allowing integration of
circulating immune cells, connective tissues cells, and complex
microbiome, which could serve as meaningful alternatives to
many types of animal testing (Figure 1). These devices may be
lined with either primary cells, iPS cells, or cells isolated from
patient-derived organoids. Importantly, while organoids may be
maintained in perfusion cultures, the advantage of microfluidic
Organ Chips is that dynamic fluid flow is delivered through an
endothelium-lined channel as it is in living organs, and hence nu-
trients, wastes, and drugs are transferred back and forth across
the endothelial interface with surrounding tissues in the chip as
occurs in vivo.
Many Organ Chip designs have been explored,[13] however, a
common embodiment is a small microfluidic device the size of a
computer memory stick composed of an optically clear flexible
polymer, such as poly-dimethylsiloxane (PDMS), that contains
two linear hollow channels each less than 1 mm wide that are sep-
arated by a thin porous membrane made of the same clear flexible
material (Figure 1).[14] By coating the membrane with ECM, it is
possible to culture and differentiate organ-specific epithelial cells
on one side of the membrane while growing endothelium from
the same organ on the other. Culture medium or whole blood

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can be flowed through the endothelium-lined microchannel to
mimic vascular perfusion, and medium can be flowed through
the epithelial channel as a form of interstitial fluid (e.g., in Liver
Chips) or the channel may be filled with air to create an air–liquid
interface (ALI) (e.g., in Lung Chips). Immune cells, either cir-
culating (Figure 1) or resident, as well as connective tissue cells
(e.g., fibroblasts, astrocytes), may be incorporated into these de-
vices as well. In addition, by applying cyclic suction to full height
side chambers on the either side of the central microchannels
in some devices, it is possible to rhythmically stretch and relax
the side walls and attached porous membrane with the adher-
ent tissue–tissue interface (Figure 1), thereby mimicking organ-
relevant motions, such as breathing in the lung or peristalsis in
the intestine.
Importantly, many different Organ Chips have been shown to
recapitulate human physiology, disease states, host–microbiome
interactions, and responses to clinically relevant drug and radi-
ation exposures with a level of fidelity that is as good or better
than animal models. For example, a Lung Alveolus Chip lined
by human A549 lung alveolar epithelial cells and pulmonary vas-
cular endothelium that experienced cyclic breathing movements
reproduced many features of human lung physiology including
gas exchange and responses to bacteria, inflammatory cytokines,
and airborne nanoparticulates introduced into the alveolar space,
as well as revealing previously unknown effects of physiological
breathing on these responses.[14] The same chip mimicked drug
toxicity-induced pulmonary edema observed in human patients
treated with the cancer drug interleukin-2 (IL-2) at a clinically
relevant dose and over the same time frame, in addition to re-
vealing that physiological breathing motions were required for
this response while immune cells were not.[15] Importantly, this
work led to the identification of a potential therapeutic (TRPV4
ion channel inhibitor) and helped to enable its entry into human
clinical trials.
A later version of the Lung Alveolus Chip lined with pri-
mary human lung alveolar cells and pulmonary microvascu-
lar endothelium that supported flowing whole human blood
through the device faithfully recapitulated organ-level contri-
butions to platelet–endothelial dynamics and inflammation-
induced thrombosis.[16] Using this chip, it was discovered that
bacterial lipopolysaccharide (LPS) endotoxin stimulates intravas-
cular thrombosis via activation of the alveolar epithelium, rather
than acting directly on endothelium. In addition, this device was
leveraged to deliver a potential adenoviral vector-based gene ther-
apy encoding a TRPV4 inhibiting peptide directly to the lung
alveolar cells (i.e., through the airspace of the epithelial channel)
while experience breathing motions, and this was able to sup-
press pulmonary vascular leakage.[17] Thus, in addition to mim-
icking human responses to pathogens, toxins, drugs, and gene
therapies, these Organ Chip studies led to new insights into the
cellular and molecular basis of pathophysiology due to their abil-
ity to enable independent control of cell composition, chemical
factors, fluid flow, and mechanical forces, which is not possi-
ble using either conventional cell culture, organoids, or animal
models.
Human Lung Airway Chips lined by primary human bron-
chiolar epithelium grown under an ALI in one microchan-
nel with an underlying microvascular endothelium in another
also can reconstitute the goblet cell hyperplasia, increased
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cytokine production, and suppressed ciliary function of asthmat-
ics when stimulated with interleukin-13.[18] When these same
Airway Chips were lined with epithelial cells from patients with
chronic obstructive pulmonary disease (COPD), they recapitu-
lated the selective cytokine hypersecretion and enhanced recruit-
ment of circulating human neutrophils that are flowed through
the endothelium-lined vascular channel, as well as clinical exac-
erbation by exposure to viral and bacterial toxins, which are seen
clinically. This inflammatory disease model was also used to de-
tect synergistic effects of lung endothelium and epithelium on
cytokine production, identify potential biomarkers of COPD exac-
erbation, and measure responses to anti-inflammatory drugs that
suppress cytokine-induced recruitment of circulating immune
cells. Because the model incorporates dynamic fluid flow, it was
possible to mimic recruitment of circulating immune cells and
their inhibition by anti-inflammatory drugs more effectively than
static Transwell cultures that contained the same tissue–tissue
interface.
Furthermore, by connecting the Lung Airway Chips lined by
primary human epithelial cells to a cigarette smoking robot that
breathes whole cigarette smoke in and out of the air channel of
the chips, it was possible to carry out matched comparative anal-
ysis of the same patients before and after smoke exposure. This
led to the identification of ciliary abnormalities, as well as COPD-
specific cytokine and transcriptomic signatures (Figure 2), that
closely matched those detected in prior clinical studies in smok-
ers who were otherwise healthy.[19] Given the anatomic and phys-
iological differences between species, these studies could not be
done as effectively in animal models, and particularly not in mice
given their small size.
Most recently, an influenza virus infection model was devel-
oped using the Lung Airway Chip, which replicates the virulence
of different viral strains (e.g., H1N1 vs H3N2 and H5N1) and en-
ables quantification of host cytokine and immune cell responses,
as well as mimicry of clinical responses to antiviral therapies and
spontaneous viral evolution in response to drug exposure that
is enabled by human-to-human transmission.[20,21] Microfluidic
Airway Chips are now being used to model human lung infection
by SARS-CoV-2 virus in vitro, and to repurpose existing FDA-
approved drugs as potential COVID-19 therapeutics.[21,22] Impor-
tantly, it is extremely difficult to develop animal models of viral
infection, and none replicate human host responses as well as
these Organ Chip models that incorporate highly differentiated
human lung cells grown under an ALI while exposed to dynamic
fluid flow at their base.
The same chip design has been used to model the responses
of human intestine to various stimuli in ways that are com-
parable to, or more accurate than, animal models. Intestine
Chips that were initially lined by human Caco-2 intestinal ep-
ithelial cells,[23–25] and later by primary cells isolated from hu-
man patient-derived duodenal or colon organoids along with pri-
mary human intestinal microvascular endothelium,[26,27] have
been shown to exhibit histological appearance similar to that ob-
served in vivo (e.g., villi-like structures in duodenum) (Figure 3A)
and high levels of goblet cell differentiation (Figure 3B), as well
as improved intestinal barrier function, enhanced drug metabo-
lizing activities, and more mucus production than static cultures.
Interestingly, when transcriptomic analysis was carried put, the
Small Intestine Chips more closely resembled living duodenum
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Figure 2. Human Lung Airway Chip recreates airway epithelial architecture and replicates patient responses to cigarette smoke in vitro. A) Still image
captured from a video recording from the side of cilia beating on the apical surface of the differentiated airway epithelium on-chip. B) Scanning electron
micrograph of the apical surface of the airway epithelium formed on-chip showing ciliated cells (blue) and non-ciliated cells (brown). C) Graph showing
changes in interleukin 8 (IL-8) secretion in Lung Airway Chips lined with epithelial cells isolated from normal or COPD patients, with or without exposure
to whole cigarette smoke for 75 min (smoking) (**p < 0.01). D) Heatmap comparing expression of 29 genes associated with cellular oxidation–reduction
in lung airway epithelial cells obtained from small airways of different normal human smokers compared with samples obtained from three Lung Airway
Chips that were exposed to whole cigarette smoke on-chip. Note that the patterns of induced and suppressed genes in the Airway Chip mimic those
seen in human patients. (B) Reproduced with permission.[18] Copyright 2016, Springer Nature. (C,D) Reproduced with permission.[19] Copyright 2016,
Cell Press.

in vivo than the organoids from which they were derived (Fig-
ure 3C).[26] Moreover, the Colon Chips lined by organoid-derived
cells spontaneously accumulate a mucus layer with the same im-
penetrable and penetrable layers, and the same thickness, as is
observed in human colon, whereas this is not observed in the
organoids cultured under the same conditions without flow.[27]
Mucus accumulation in the Colon Chips also can be studied non-
invasively over time on-chip using live imaging, which is not pos-
sible in other cell cultures or animal models.
Small Intestine Chips lined by cells isolated from patient-
derived organoids have been used to study induction of CYP450
metabolizing enzymes that can complicate the development of
new drugs, and provide insight into the molecular and cellular ba-
sis of drug–drug interactions that can cause changes in drug PK,
safety, and efficacy.[28] Relative to animal models, Small Intestine
Chips better mimic infection by enteric viruses[29] and intesti-
nal injuries induced by acute exposure to 𝛾-radiation (including
dosage sensitivities and responses to countermeasure therapies)
exhibited by humans as well.[30]
Additional Small Intestine Chip studies showed that probi-
otic and antibiotic therapies can prevent villus injury induced by
pathogenic bacteria, and this led to identification of four proin-
flammatory cytokines (IL-8, IL-6, IL-1𝛽, TNF-𝛼) elicited by LPS
endotoxin and human immune cells that are necessary and suffi-
cient to compromise intestinal barrier function.[25] Interestingly,
use of mechanically active Organ Chips also revealed that ces-
sation of mechanical peristalsis-like motions stimulates over-
growth of bacteria,[26–28] as seen in patients with ileus and in-
flammatory bowel disease (IBD). Moreover, similar results were
obtained using a human Colon Chip, which showed that
peristalsis-like mechanical deformations influence infection by
the human pathogen Shigella.[31] Finally, additional complexity
has been integrated into Intestine Chip models by establishing
a hypoxia gradient across the tissue–tissue interface, which en-
ables extended co-culture of human intestinal epithelium in di-
rect contact (via its natural overlying mucus layer) with complex
living human gut microbiome containing over 200 different types
of microbial strains over multiple days in vitro.[32] This is yet an-
other advantage of human Organ Chips over static MPS cultures,
organoids, and animal models, as they do not permit long-term
human microbiome co-cultures.
Yet another example where Organ Chip experiments provide
advantages over animal studies relates to modeling the blood–
brain barrier, which has distinct permeability properties and

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Figure 3. Human Intestine Chips lined by cells isolated from patient-
derived organoids exhibit differentiated structures and functions that
closely resemble those displayed by living intestine in vivo. A) Differen-
tial interference microscopic image (top) and immunofluorescence mi-
croscopic image of F-actin staining in green (bottom) of vertical sec-
tions through an Intestine Chip showing villus protrusions formed by
primary intestinal (duodenal) epithelium cultured on-chip for 12 days.
B) Immunofluorescence microscopic image of a vertical section through
a human Colon Chip showing a high polarized epithelium with basolat-
eral adherens junctions labeled with E-cadherin (green) and brush border
stained for F-actin (white) restricted to the apical regions, Hoechst stained
nuclei (blue) localized at the cell base, and goblet cells stained for Muc2
(magenta). C) A curated heatmap showing gene expression profiles in the
mechanically active Intestine Chip (with fluid flow and cyclic stretching to
mimic peristalsis-like motions) lined by cells from patient-derived duode-
nal organoids, the duodenal organoids from which the cells were derived,
and duodenum in vivo. Note that the expression of pattern of the Intestine
Chip more closely resembles native intestine compared to the organoids.
(A,C) Reproduced with permission.[26] Copyright 2018, Springer Nature.
(B) Reproduced with permission.[27] Copyright 2020, Elsevier Inc.
transport functions in humans. Human Blood–Brain Barrier
Chips have been developed that are lined by iPS cell-derived
human brain microvascular endothelium interfaced with either
primary human brain pericytes and astrocytes[33] or human iPS-
derived astrocytes and neurons,[37] which reconstitute the high
permeability restrictions of the human barrier. When differen-
tiated appropriately, the iPS cell-derived endothelium expresses
high levels of human-specific efflux pumps and tight junction
proteins, which enables recapitulation of differential transport
and selective transcytosis of antibodies and peptides, as well as ex-
tracellular vesicles, that have been observed in vivo.[33–35] Blood–
Brain Barrier Chips containing iPS-derived human neurovascu-
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lar cells isolated from patients also were able to recreate disease-
specific transporter deficiencies and barrier disruption.[34] More-
over, when the vascular lumen was perfused with whole blood,
the engineered blood–brain barrier protected neural cells from
plasma-induced toxicity and accurately predicted brain perme-
ability of drugs. These observations raise the possibility that hu-
man Blood–Brain Barrier Chips may provide a more physiologi-
cally relevant way to develop shuttle molecules for delivery of ther-
apeutic antibodies and other brain-targeting therapeutics than
mouse or non-human primate models, which are used today.
Different useful functionalities and biological mimicry has
been demonstrated using a human Bone Marrow Chip that main-
tains differentiation and maturation of multiple blood cell lin-
eages over 1 month by co-culturing CD34+ cells and marrow-
derived stromal cells in a fibrin gel in one channel interfaced with
human microvascular endothelium in the other (Figure 4A).[36]
This chip reproduces marrow toxicities induced by exposures to
clinically relevant doses of anti-cancer drugs (Figure 4B) or ioniz-
ing radiation, as well as bone marrow recovery after drug-induced
myelosuppression, whereas other preclinical models do not.
Importantly, using this microfluidic chip, it was possible to
recapitulate both drug exposure profiles that were previously
measured in PK studies in human patients (Figure 4C) and as-
sociated regimen-specific myeloerythroid toxicity responses pre-
viously measured in those same patients (Figure 4D). Equally
important, characteristic hematopoietic defects of a rare genetic
disorder (Shwachman–Diamond syndrome) were recapitulated
on-chip by incorporating cells into the Bone Marrow Chips that
were isolated from patients, and this work led to new insight in
the underlying pathogenesis of these disease. The ability of this
Bone Marrow Chip to replicate drug PK-dependent effects, clin-
ically relevant radiation sensitivities, and a rare human disease
phenotype using patient-specific cells under these dynamic mod-
eling conditions, is a clear example of why human Organ Chips
should be considered as alternatives to animal models.
Human Organ Chips also have been used to create “ortho-
topic” Cancer Chip models by growing tumor cells in organ-
relevant contexts, rather than relying on injecting human tu-
mor cells into their organs of origin in mice. For example, when
human non-small-cell lung cancer (NSCLC) cells that arise at
the bronchiolar–alveolar interface but grow preferentially within
alveoli in patients, were cultured in human Lung Alveolus and
Airway Chips, they recapitulated these organ microenvironment-
specific behaviors by proliferating rapidly in the former and ex-
hibiting tumor dormancy in the latter.[37] In addition, these mod-
els replicated differences in responses to tyrosine kinase inhibitor
(TKI) therapies observed in human patients, and revealed that
breathing motions can influence lung cancer cell growth and in-
vasion, as well as therapeutic responses. Again, these insights
would not be possible using animal models.
In another cancer study, a microfluidic Organ Chip model of
the human bone perivascular niche containing vascular cell net-
works and marrow-derived mesenchymal stem cells within a na-
tive bone matrix with controlled flow hemodynamics and oxy-
gen gradients revealed that human breast tumor cells exposed
to interstitial flow persist in a dormant state on-chip that is as-
sociated with increased drug resistance.[38] A perfused model of
metastatic tumor seeding in liver that incorporates human hepa-
tocytes, nonparenchymal liver cells, and breast cancer cells, and
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which was instrumented with oxygen sensors and micropumps
that enabled diurnal variations in nutrient and hormone profiles,
was similarly used to recreate the human-specific metastatic mi-
croenvironment and probe the paracrine effects between liver
cells and metastatic tumor cells.[39] More recently, a microfluidic
model containing a 3D collagen gel populated by human liver
cancer cells in one channel was challenged with human T cells
engineered to express tumor-specific T cell receptors in an ad-
jacent channel while modulating oxygen levels and inflamma-
tory cues to assess the preclinical efficacies of adoptive T cell
immunotherapies in an immunosuppressive environment.[40]
These types of studies can provide insight into human-specific
cancer and immune cell responses that would not be possible
with animal models.
Organ Chips with different fluidic designs have produced ad-
ditional evidence showing that this technological approach can
be used with primary or patient-derived cells to gain insight into
human-relevant cellular and molecular mechanisms of patho-
genesis, as well as drug actions, more effectively than animal
models. For instance, a human Organ Chip model of the kid-
ney proximal tubule was combined with quantitative systems
pharmacology-based computational models to enable in vitro-
to-in vivo translation (IVIVT) of experimental results for the
biomarker kidney injury molecule-1 (KIM-1), which predicted
human drug-induced renal toxicities, and helped to identify fa-
vorable dosing regimens.[41] The same approach was leveraged
to uncover a unique mechanism by which an FDA-approved an-
tibiotic drug (polymyxin) induces kidney injury, and to identify
related chemical analogues that do not induce this toxicity.[42] An-
other Kidney Chip was used to model viral infection of the kidney
distal tubule and how it alters sodium reabsorption, which can
lead to serum electrolyte abnormalities.[43] Finally, a microfluidic
model of the kidney glomerulus lined by human iPS cell-derived
kidney podocytes interfaced with kidney microvascular endothe-
lium and exposed to cyclic mechanical deformations (mimicking
changes in blood flow with each beat of the heart) reconstituted
differential clearance of albumin and inulin via glomerular filtra-
tion, as well as albuminuria and podocyte injury induced by the
cancer drug adriamycin.[44]
While most work in this field has focused on creating Organ
Chips lined by human cells, species-specific Organ Chips also
have been made that, for example, can replicate the much higher
susceptibility of humans to infection by enterohemorrhagic Es-
cherichia coli (EHEC) compared to mice.[45] Because fluids are
continuously flowed through these chips, it was possible to carry
out metabolomic analysis in this study using a Colon Chip, which
led to the identification of specific chemical metabolites that are
responsible for this different susceptibility to EHEC infection.
Rat, dog, and human Liver Chips containing primary hepato-
cytes, liver sinusoidal endothelium, Kupffer cells and stellate cells
also have been created that replicate species-specific hepatotoxi-
cities induced by multiple drugs (Figure 5).[46] This is an impor-
tant finding because preclinical rat and dog liver toxicity studies
are key requirements for regulatory approval, and they can of-
ten produce conflicting results, leading to confusion or removal
of good drugs from the pipeline. These results suggest that hu-
man Organ Chips offer a potential way to cross-validate these re-
sults between animals and humans early in the drug develop-
ment process. These animal Organ Chips also might serve as re-
placements for animal validation studies, for example, in veteri-
nary applications or be used to benchmark in vitro results versus
findings obtained in past animal experiments.
More sophisticated microenvironmental control can be inte-
grated into Organ Chips because of their microfluidic design. For
example, continuous oxygen zonation can be generated in Liver
Chips by regulating flow rate in the vascular and hepatic chan-
nels, which can provide insight into its contribution to human
liver physiology, toxicology, and disease progression, as well as
recruitment of circulating immune cells.[47] By populating per-
fused Liver Chips with freshly cryopreserved hepatocytes from
five different human donors, it also has been possible to quan-
tify metabolic clearance rates for multiple drugs, and this led to
the finding that albumin, urea, lactate dehydrogenase, and cy-
tochrome P450 mRNA levels can serve as predictors of these
pharmacologically relevant responses.[48] In addition, by overlay-
ing a computational, population-level, physiologically based PK
model, in vitro data from this study were related to their ob-
served PK behavior in vivo, and model simulations predicted ob-
served clinical concentration-time profiles and associated popu-
lation variability in drug metabolism. The same perfused Liver
Chip also was able to recapitulate the clinical impact of an anti-
IL-6R monoclonal antibody used to treat rheumatoid arthritis on
small molecule drug PK, and it was shown to be mediated by
modulation of cytochrome P450 enzyme activities.[49]
Another example where Organ Chips have been shown to
be a valuable alternative to animals is in ophthalmologic drug

Figure 4. Prediction of clinically observed hematotoxicities at patient-relevant drug exposures using a human Bone Marrow Chip. A) Left, schematic of
bone and insert showing normal human marrow histology. Left middle, schematic of the human Bone Marrow Chip at the time of seeding, showing
dispersed CD34+ progenitor cells and bone marrow stromal cells in an ECM gel filling the top channel, and a vascular endothelium beginning to line
the adjacent channel. Right middle, within 2 weeks of culture, the endothelium covers all four sides of the lower channel, creating a vascular lumen, and
the CD34+ cells undergo expansion and multilineage differentiation. Right, immunofluorescence view of a cross-section through the ECM gel grown
on-chip for 14 days (yellow, megakaryocyte lineage; magenta, erythroid lineage; blue, neutrophil and other hematopoietic lineages; EC, endothelial cell).
B) Effects of treating the Bone Marrow Chips, suspension cultures, and static ECM gel co-cultures for 48 h with various doses of the cancer drug, 5-
fluorouracil (5-FU) on total cell number (left) versus only neutrophil lineage cells (right)(***p < 0.001). The range of patient plasma 5-FU concentrations
for a 2 day infusion that are known to cause myelosuppression is indicated in orange. Note that the Bone Marrow Chip replicates this sensitivity to a
clinically relevant dose exposure whereas the other in vitro models do not. C) Graphs showing that dynamic changes in concentrations of the cancer drug
AZD2811 measured by mass spectrometry in chip outlet (circles), which were used to fit PK models of Bone Marrow Chip drug exposure (black line) for
2 and 48 h infusions. Note that these drug exposure profiles that were generated in the microfluidic Bone Marrow Chip closely resembled plasma levels
of AZD2811 in vivo (grey), which were simulated for an average patient at a range of clinical doses based on the known PK characteristics of AZD2811.
D) Graphs showing the effects of infusing the Bone Marrow Chips with varying doses of AZD2811 for 2 versus 48 h; total neutrophil (blue) and erythroid
(red) cell numbers on day 12 are shown (***p < 0.001). Note that the clinical observation of unusual regimen-specific neutropenia and anemia with 2
h dosing, but only neutropenia with 48 h dosing, was replicated on-chip. Reproduced with permission.[36] Copyright 2020, Springer Nature.

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www.advancedsciencenews.com www.advancedscience.com

Figure 5. A human Liver Chip recapitulates species-specific drug toxicities in rat, dog, and human. A) Schematic of the Liver Chip that contains an upper
parenchymal channel lined by primary rat, dog, or human hepatocytes grown in ECM sandwich, while species-specific liver sinusoidal endothelial cells
(LSECs), Kupffer cells, and stellate cells are cultured on the opposite side of the same membrane in the lower vascular channel. B) Previously observed
species-specific effects of the drug bosentan on albumin secretion were recreated in microfluidic human, dog, and rat Liver Chips (Chip, open circles)
whereas this was not replicated in static hepatocyte sandwich monocultures (Plate, closed triangles). Reproduced with permission.[46] Copyright 2019,
American Association for the Advancement of Science.

development where results from animal experiments often do
not translate to humans. Recently, a Retina Chip was created
that is lined by retinal organoids derived from human iPS cells
that form complex stratified retinal tissues containing more than
seven different retinal cell types, and that permits physiological
interactions between mature photoreceptor segments with reti-
nal pigmented epithelium.[50] This chip reproduced the retino-
pathic side effects of the anti-malaria drug chloroquine and the
antibiotic gentamicin in vitro, and now offers a preclinical hu-
man model to study drug efficacy and toxicities in a way that is
not possible in animal models.
Perhaps the clearest argument against using animal models
comes from studies that show human Organ Chips can repli-
cate pathophysiological responses observed in humans that were
never detected in preclinical animal studies. One example is a
human Kidney Chip model of the proximal tubule that can repli-
cate toxicities induced by cisplatin through a human-specific Pgp
efflux transporter that is not expressed in animals.[51] Another is
the demonstration that a human Blood Vessel Chip reproduces
thrombotic toxicities in response to treatment with a therapeu-
tic monoclonal antibody that caused deaths in human clinical
trials even though this toxicity was not observed in preclinical
animal studies.[52] Finally, one challenge that is difficult for an-
imal models is to faithfully mimic complex function of the hu-
man immune system, such as vaccination responses. However,
recent studies suggest that this also may be possible using a hu-
man Organ Chip that supports self-assembly of human blood-
derived B and T lymphocytes into germinal center-like lymphoid
follicles.[53] Also, the human Bone Marrow Chip supports con-
tinued production of multiple blood cell lineages in vitro, as well
as spontaneous intravasation of more differentiated blood cells
into the endothelium-lined vascular channel of these microflu-
idic chips,[36] raising the possibility of creating fluidically linked
single or multiple Organ Chip systems that incorporate all cells
and tissues derived from the same patient, including circulating
immune cells.
3.4. Multi-Organ Chip Models
One of the greatest advantages of microfluidic Organ Chips
over static engineered MPS and organoids is that multiple chips
can be linked fluidically to model multi-organ physiology and
even create human “Body-on-Chips” models that can be used for
PK studies. Two microfluidic human Blood Brain Barrier Chips
(lined by primary brain microvascular endothelial cells interfaced
with human astrocytes and pericytes) were fluidically coupled via
their interstitial (cerebral spinal fluid) channels before and af-
ter a chip containing primary human brain neuronal networks
(Figure 6A–C) to model drug influx across the human blood–
brain barrier, permeation into the brain parenchymal compart-
ment, and efflux across the barrier.[54] This model of the hu-
man neurovascular unit effectively mimicked the barrier open-
ing properties of the psychoactive drug methamphetamine. It
also permitted independent metabolomic analysis of each of the
different fluidic compartments of the neurovascular unit, which
revealed previously unknown metabolic coupling between the
brain microvascular endothelium and neurons that would be im-
possible to do in animal studies.
In a separate study, a microfluidic multi-organ model con-
taining gut, liver, and circulating Treg and Th17 cell compo-
nents was used to model the contributions of microbiome and
liver metabolism to IBD.[55] This work revealed that microbiome-
derived short-chain fatty acids can influence IBD severity based
on the involvement of effector CD4 T cells. Multi-Organ Chip
systems also have been used to model preclinical drug toxicities
and metabolic conversion,[56] as well as sequential multi-organ
metabolism of drugs and xenobiotics that can impact therapeu-
tic action and side effects.[57] In the latter study, Organ Chips
representing the jejunum, liver, and kidney (the major absorp-
tion, metabolism, and clearance organs) were evaluated, as well
as skeletal muscle and neurovascular models, and organ-specific
ADME processing of various drugs was shown to be consistent
with human clinical data.
It has even been possible to study organism-level, hormonal
cross-talk, such as between insulin production in pancreas and
glucose regulation in liver by fluidically coupling two microflu-
idic chips lined by human pancreatic islet microtissues and liver
spheroids.[58] A functional hormonal feedback loop between the
human liver and insulin-secreting islet tissues was demonstrated
in this study, which suggests that it might be useful for study-
ing diabetes in humans. Even more impressively, a functioning
female reproductive system that can be used to study the hu-
man 28-day menstrual cycle and pregnancy-associated hormonal
loops has been successfully recreated in vitro by fluidically cou-
pling organ modules for the ovary, fallopian tube, uterus, cervix,
and liver.[59]

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Figure 6. Multi-Organ Chip model of the human neurovascular unit. A) Schematic of the influx Blood–Brain Barrier Chip containing brain microvascular
endothelium (magenta) in its vascular channel separated by a porous membrane from brain astrocytes (blue) and pericytes (yellow) in the parenchymal
channel through which medium mimicking cerebral spinal fluid (CSF) flows. The CSF fluid is transferred to a similar channel within a Brain Chip that is
separated by a porous membrane from a channel containing cultured human brain neuronal cells (green) and astrocytes (blue), and from there to the
parenchymal channel of a second efflux Blood–Brain Barrier Chip. Medium mimicking blood is flowed separately through the lower vascular channel. B)
3D confocal microscopic reconstruction of the Blood–Brain Barrier chip viewed from the side showing a continuous endothelium stained for VE-cadherin
(purple) forming a lumen in the lower vascular channel, as well as pericytes (F-actin, yellow) and astrocytes (GFAP, blue) on the top surface of the porous
membrane in the upper channel. C) Confocal fluorescence microscopic view of networks of human brain neurons (𝛽-III-tubulin, green) and astrocytes
(glial fibrillary astrocytic protein, GFAP, blue) in the lower compartment of the Brain Chip. Reproduced with permission.[54] Copyright 2018, Springer
Nature.

An outstanding example of a capability that Organ Chip tech-
nology offers that animal models do not relate to the modeling of
human drug PK properties using human Body-on-Chips mod-
els that can maintain up to ten different human Organ Chips
when fluidically coupled for 1 month in vitro.[60,61] PK studies
have been carried out in many multi-Organ Chip models by
transfering fluids between them, and analyzing drug levels and
metabolism using mass spectrometry.[59,62–65] In some studies,
qualitative predictions of drug toxicity responses have been made
using PK and pharmacodynamic models with coupled Organ
Chips.[60,66–69] However, in these studies, the medium contain-
ing drug flowed directly from one parenchymal tissue type to an-
other without passing through the endothelium, which is crucial
for defining drug PK behavior in vivo, and thus, the physiologi-
cal accuracy of these results is limited. However, more recently,
it has been possible to quantitatively predict drug PK parame-
ters (e.g., Cmax , T1/2 ) measured in human patients by applying
physiologically based PK modeling to experimental data obtained
with a human Body-on-Chips platform that coupled fluid flows
between endothelium-lined vascular channels of Liver, Kidney,
and Intestine Chips to model first-pass drug ADME behaviors
(Figure 7A).[61,70] This multi-Organ Chip platform also incorpo-
rates an arterio-venous fluid mixing reservoir so that drug con-
centrations within the vascular channels of all chips can be mea-
sured by sampling this shared compartment, much like taking
venous blood samples in a patient. Quantitative PK predictions
were demonstrated for two different drugs (cisplatin and nico-
tine) that were administered via two different routes (intravenous
and oral, respectively) (Figure 7B), and predictions of cisplatin
pharmacodynamics obtained in a fluidically linked Bone Marrow
Chip also matched previously reported patient data.[70] Together,
these studies show how human Body-on-Chips approaches can
enable quantitative IVIVT of human PK and pharmacodynamic
parameters, as well as predict drug ADME properties and toxici-
ties, which is not currently possible with animal models.
4. Advantages and Disadvantages of Organ
Chip Models
From this analysis, it appears that human Organ Chips can
provide much of the organ-level information that Reviewer 3 is
seeking when he or she requests additional animal validation
studies, and even provide useful insight into multi-organ physiol-
ogy. The presence of dynamic fluid flow, tissue–tissue interfaces,
and physiological mechanical cues enhances the fidelity of cell
differentiation and increases expression of tissue-specific func-
tionalities on-chip relative to conventional cultures, static MPS,
and even organoids.[14,18,23,26,27,33,36,44,46,71] Tissues in Organ Chips
also can be probed using virtually any type of analytic technique
that is used in other in vitro models or animal studies, including
methods leveraging high resolution microscopy, flow cytometry,
transcriptomics, proteomics, metabolomics, and histological
analysis, and as recently demonstrated, automated high content
confocal imaging with fluorescent reporters.[72] In addition, be-
cause Organ Chips can be instrumented with in-line electrical,
chemical, mechanical, and optical sensors (both alone and in
combination), they can incorporate real-time readouts and en-
able probes of critical cell and tissue functions, including tissue

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Figure 7. A first-pass human Body-on-Chips model that predicts human PK parameters. A) Top, photographs of the Gut, Liver, and Kidney Chips that
were fluidically coupled to each other and to an arterio-venous mixer reservoir to create the first-pass PK model in vitro. Bottom, diagrams of the different
2-channel microfluidic chips with arrows indicating the manner in which the chips and reservoir are fluidically linked to each other and to the AV reservoir
(red arrows indicate flow direction). B) Predictions of how nicotine blood concentrations will change over time for three different oral doses (different
colored dashed lines) made by a physiological PK model using experimental data obtained from the human Body-on-Chips platform compared with
previously published blood nicotine levels measured in patients who received orally administered nicotine in the form of nicotine gum (blue), pouched
chewing tobacco known as “snus” (black), or loose snus (green) at three different doses (4, 9, and 13–16 mg). Note that the experimental platform was
able to quantitatively predict these human PK behaviors. C) Graph showing changes in cisplatin concentrations in blood over time predicted by the same
PK model using data from a Body-on-Chips configuration containing linked Liver, Kidney, and Bone Marrow Chips along with an arterio-venous reservoir
for infusion periods (dotted lines) of either 1 h (black) or 3 h (blue). Note that these predictions closely match previously published blood cisplatin
levels measured in patients who received cisplatin injected intravenously over these same time periods (solid lines). Reproduced with permission.[70]
Copyright 2020, Springer Nature.

barrier integrity, neuronal cell electrical activity, oxygen uti-
lization, pH, molecular transport, and multiple other relevant
measures of differentiation state and viability.[10,11,32,47,73–79] The
ability to control the contents of different parallel flow paths in-
dividually enables regulation of oxygen and chemical gradients
across tissue–tissue interfaces as well, which permits stable co-
culture of living human tissues in direct contact with living com-
plex microbiome as in the human intestine,[32] as well as recre-
ation of oxygen zonation patterns in organs, such as the liver.[47]
All of these measurements can be carried out under dynamic
flow conditions as occurs in vivo, and many would be difficult, if
not impossible, to carry out in animal studies or even in human
trials.
Organ Chips offer a potentially useful alternative to animal
testing, but they cannot replicate many complex functions and
responses that are still better studied in animals. The most ob-
vious examples relate to studies focused on cognitive functions
and complex organismal behaviors, which remain beyond the ca-
pabilities of models that rely on cultured cells. Similarly, while
brain neurons have been integrated into some microfluidic Or-
gan Chips,[34,54] peripheral neurons have not; this is an area that
clearly needs greater exploration before animal studies can be re-
placed. Quantitative analysis of the effects of large-scale forces
and deformations on large organs (e.g., long bones, spinal disks,
whole joints), or of the contributions of macroscale 3D architec-
ture to host responses (e.g., infections of the lung apex vs base),
also cannot be studied using Organ Chip technology, although
some simplified models can be developed to study some of the
underlying cellular mechanisms (e.g., local effects of compres-
sion on bone or cartilage tissue). While mimicry of some hor-
monal axes has been accomplished using microfluidic Organ
Chips, such as the reproduction of the menstrual cycle in vitro,[58]
it is difficult to model temporal variations in many different hu-
man hormones simultaneously as can occur in vivo; moreover,
many bloodborne regulatory factors that could be important for
biological regulation are still unknown. But because of flow con-
trol, it is possible to replicate temporal changes in hormone levels
in Organ Chips for hormones that have been well characterized
in vivo.
The lack of fat tissues that can absorb many drugs and
lipophilic compounds in vivo also could produce abnormal
results in experiments studying how drugs distribute in

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www.advancedsciencenews.com
multi-Organ Chip models. Some materials that are used to con-
struct Organ Chip devices, such as PDMS, can absorb lipophilic
drugs and hence, this could similarly interfere with drug stud-
ies; interestingly, however, by quantifying the level of absorption
into these materials (e.g., using mass spectrometry), this fea-
ture can be used to mimic fat absorption in human Body-on-
Chips modeling.[61,70] Another advantage human Organ Chips
could potentially provide relative to animal models is the abil-
ity to replicate human-specific immune and inflammatory re-
sponses that are not seen in other species. Circulating immune
cells have been incorporated into multiple human Organ Chip
models[9,14,18,21,25] and complex vaccination responses have been
replicated on-chip.[53] But to meet this goal of fully recapitulat-
ing human immune responses in vitro, it will be necessary to
incorporate tissue-resident immune cells and obtain parenchy-
mal, connective tissue, endothelial, and immune cells all from
the same donor.
Another challenge that the Organ Chip field faces relates to
cell sourcing. While established cell lines generally fail to repli-
cate tissue-specific functions with high fidelity, primary human
cells and organoids can display significant donor-to-donor vari-
ability. The quality of commercially available primary cells (e.g.,
doubling time, passage number, differentiation potential) also
can vary greatly between different suppliers and even between
different lots from the same company. However, these issues can
be managed by establishing protocols for “qualifying” each cell
source in terms of the consistency and quality of their growth and
differentiation potential before initiating critical experiments.
Moreover, inter-donor variability, while frustrating relative to
working with established cell lines or inbred animals, is much
more clinically relevant as it mimics the diversity of responses
seen in clinical trials and broader human populations. This chal-
lenge can be overcome by repeating studies with cells from mul-
tiple donors, and exploring whether male and female cell sources
produce different results can lead to helpful insights as well. In-
terestingly, however, studies with Lung Airway Chips[18,19] and
Bone Marrow Chips[36] suggest that results can be quite repro-
ducible between different donors.
Finally, it is important to note that Organ Chips do not mimic
the function of a whole organ; rather, they are effectively living 3D
cross-sections of a major functional subunit of an organ, such an
alveolus, airway, glomerulus, or proximal tubule. So before any
animal model can be replaced, the Organ Chip must be designed
so that it contains the key features of the relevant organ unit that
is the key site of this activity, while minimizing system complex-
ity. For example, while a Lung Alveolus Chip containing only alve-
olar epithelium, endothelium, and immune cells might be used
to replace an animal model of pulmonary edema that is focused
on preventing pulmonary vascular leakage, it would need to be
modified by adding additional connective tissue cells to replace
an in vivo pulmonary fibrosis model. Thus, the goal is not to use
Organ Chips to replace animal models in a generic way; rather, it
is to replace one specific type of animal model at a time. Similarly,
human Body-on-Chips platforms need to be designed to model
the organism-level properties of interest, and recent studies sug-
gest that meaningful results can be obtained using this approach
that may be even more useful than those obtained from animal
studies, as demonstrated by the ability to quantitative predict hu-
man drug PK parameters using a first pass intestine–liver–kidney
Adv. Sci. 2020, 2002030 2002030 (12 of 15)
www.advancedscience.com
model,[70] which could potentially accelerate clinical trials. This
inability of a single Organ Chip to model all facets of that organ’s
function, and the need to modify both single and multiple Organ
Chip systems based on the questions being posed, are challenges
that animal models do not have. Furthermore, regardless of the
device design, all chips must be experimentally validated in terms
of their ability to recapitulate the key physiological or pathophysi-
ological properties of the particular organ, disease state, or multi-
organ coupling that is the focus of the study, and to do it in a
consistent and robust manner.
5. Conclusion
This article began with a simple question: does it make sense
for reviewers to demand animal validation studies before being
willing to fund grants or share articles with the larger scientific
community, and if not, is there a better alternative? As described
above, the problem mainly lies in the fact that many animal mod-
els are physiologically irrelevant when considering human dis-
ease, and thus, demanding use of a poor animal model for the
sole sake of “satisfying Reviewers” should be discouraged. Given
recent advances in human Organ Chip technologies, I suggest
that these new forms of human experimentation in vitro provide
more physiologically and clinically relevant preclinical models for
studying both pathophysiology and pharmacological responses
than many animal studies, and they should be considered for this
purpose in the future. Organoids and other MPS models that of-
ten can be carried out at a higher throughput than microfluidic
Organ Chips also have a very important role to play, particularly
in studies focused on cellular and molecular mechanisms of tis-
sue regulation and drug action. Organ Chips can be used for this
purpose as well; however, their greatest novelty comes from their
ability to reconstitute dynamic vascular perfusion, tissue–tissue
interfaces, and organ-specific mechanical cues that are impor-
tant for in vivo-like functions, and to provide an ability to se-
lectively add complexity to these models in the form of soluble
factors and different cell types (e.g., epithelial, endothelial, con-
nective tissue, immune, nerve, muscle, commensal microbes).
Thus, Organ Chips are a form of synthetic biology that allows
one to build artificial models with defined composition at the cell,
tissue, and organ level, and thereby, to identify key cellular and
molecular contributors to human physiology and pathophysiol-
ogy. For example, it would be extremely difficult, if not impossi-
ble, to identify the individual contributions of microbiome, me-
chanical cues, and specific cytokines to pathophysiology at the or-
gan and multi-organ level using animal models, whereas this has
been easily done using Organ Chips. At the very least, manuscript
and grant reviewers should first consider non-animal alterna-
tives, such as human Organ Chips, and second, whether or not a
fully validated animal model is available.
Importantly, this article is not an argument to stop all animal
testing, and to carry out all validation studies using human Or-
gan Chips instead. Clearly, we will need to continue some forms
of animal testing for many years to come in order to cross-validate
Organ Chip results with the current state-of-the-art, and to pro-
gressively convince animal researchers and regulatory scientists
of their value. And it is likely that some animal studies will be very
hard to replace (e.g., those focused on cognition and behavior).
Rather, my goal here is to initiate a conversation among members
© 2020 The Authors. Published by Wiley-VCH GmbH
www.advancedsciencenews.com
of our community, and to ask scientists who serve as reviewers to
consider the realities as they stand today. As our collective inter-
est is in human health, our goal should not be to validate results
against animals, but rather against humans, because in the end
we all know that mice are not men. Perhaps it is time for review-
ers to accept this reality.
Acknowledgements
The author thanks all of the research team members, past trainees,
collaborators, and other members of the scientific community (includ-
ing those he might have inadvertently failed to cite) for helping to es-
tablish and build the Organ Chip field. Work from the laboratory re-
viewed here was funded by DARPA (W911NF1920023, W911NF1920027,
W911NF-12-2-0036, HR0011-20-2-0040), FDA (75F40119C10098), NIH
(UH3HL141797), Cancer Research United Kingdom (C25640/A29057),
and Gates Foundation (OPP1173198, INV-002164).
Conflict of Interest
The author has the following potential conflicts: Emulate Inc., equity, con-
sulting, chair of SAB; BOA Biomedical Inc., equity, consulting, chair of SAB,
board member; Free Flow Medical Device, equity; SynDevRx, equity; Con-
sortia Rx, equity, board member; Roche, consulting; Astrazeneca, spon-
sored research; Fulcrum Therapeutics, sponsored research; Kraft Heinz,
sponsored research; and inventor of multiple patent applications.
Keywords
microfluidics, microphysiological systems, organoids, organ-on-a-chip,
preclinical studies
Received: May 29, 2020
Revised: July 22, 2020
Published online:
[1] A. Akthar, Camb. Q. Healthc. Ethics 2015, 24, 407.
[2] L. Meigs, L. Smirnova, C. Rovida, M. Leist, T. Hartung, Altex 2018, 35,
275.
[3] P. Pound, M. Ritskes-Hoitinga, J.Transl. Med. 2018, 16, 304.
[4] J. Seok, H. S. Warren, A. G. Cuenca, M. N. Mindrinos, H. V. Baker, W.
Xu, D. R. Richards, G. P. McDonald-Smith, H. Gao, L. Hennessy, C. C.
Finnerty, C. M. López, S. Honari, E. E. Moore, J. P. Minei, J. Cuschieri,
P. E. Bankey, J. L. Johnson, J. Sperry, A. B. Nathens, T. R. Billiar, M.
A. West, M. G. Jeschke, M. B. Klein, R. L. Gamelli, N. S. Gibran, B.
H. Brownstein, C. Miller-Graziano, S. E. Calvano, P. H. Mason, et al.,
Proc. Natl. Acad. Sci. U. S. A. 2013, 110, 3507.
[5] R. Franco, A. Cedazo-Minguez, Front. Pharmacol 2014, 5, 146.
[6] R. S. Herati, E. J. Wherry, Cold Spring Harb. Perspect. Biol. 2018, 10,
a031583.
[7] H. Clevers, Cell 2016, 165, 1586.
[8] K. A. Homan, N. Gupta, K. T. Kroll, D. Kolesky, M. Skylar-Scott, T.
Miyoshi, D. Mau, M. Todd Valerius, T. Ferrante, J. V. Bonventre, J. A.
Lewis, R. Morizane, Nat. Methods 2019, 16, 255.
[9] J. T. Neal, X. Li, J. Zhu, V. Giangarra, C. L. Grzeskowiak, J. Ju, I. H. Liu,
S. Chiou, A. A. Salahudeen, A. R. Smith, B. C. Deutsch, L. Liao, A. J. Ze-
mek, F. Zhao, K. Karlsson, L. M. Schultz, T. J. Metzner, L. D. Nadauld,
Y. Tseng, S. Alkahairy, C. Oh, P. Keskula, D. Mendoza-Villanueva, F.
M. De La Vega, P. L. Kunz, J. C. Liao, J. T. Leppert, J. B. Sunwoo, C.
Sabatti, J. S. Boehm, W. C. Hahn, G. X. Y. Zheng, M. M. Davis, C. J.
Kuo, Cell 2018, 175, 1972.
Adv. Sci. 2020, 2002030 2002030 (13 of 15)
www.advancedscience.com
[10] G. Wang, M. L. McCain, L. Yang, A. He, F. S. Pasqualini, A. Agarwal,
H. Yuan, D. Jiang, D. Zhang, L. Zangi, J. Geva, A. E. Roberst, Q. Ma,
J. Dian, J. Chen, D.-Z. Wang, K. Li, J. Wang, R. J. A. Wanders, W. Kulik,
F. M. Vaz, M. A. Laflamme, C. E. Murry, K. R. Chien, R. I. Kelley, G. M.
Church, K. K. Parker, W. T. Pu, Nat. Med. 2014, 20, 616.
[11] Y. Zhao, N. Rafatian, N. T. Feric, B. J. Cox, R. Aschar-Sobbi, E. Y.
Wang, P. Aggarwal, B. Zhang, G. Conant, K. Ronaldson-Bouchard, A.
Pahnke, S. Protze, J. H. Lee, L. Davenport Huyer, D. Jekic, A. Wickeler,
H. E. Naguib, G. M. Keller, G. Vunjak-Novakovic, U. Broekcel, P. H.
Backx, M. Radisic, Cell 2019, 176, 913.
[12] U. Marx, T. Akabane, T. B. Andersson, E. Baker, M. Beilmann, S.
Beken, S. Brendler-Schwaab, M. Cirit, R. David, E.-M. Dehne, I.
Durieux, L. Ewart, S. C. Fitzpatrick, O. Frey, F. Fuchs, L. G. Griffith,
G. A. Hamilton, T. Hartung, J. Hoeng, H. Hogberg, D. J. Hughes, D.
E. Ingber, A. Iskander, T. Kanamori, H. Kojima, J. Kuehnl, M. Leist, B.
Li, P. Loskill, D. L. Mendrick, et al., Altex 2020, 37, 364.
[13] S. N. Bhatia, D. E. Ingber, Nat. Biotechnol. 2014, 32, 760.
[14] D. Huh, B. D. Matthews, A. Mammoto, M. Montoya-Zavala, H. Y.
Hsin, D. E. Ingber, Science 2010, 328, 1662.
[15] D. Huh, D. C. Leslie, B. D. Matthews, J. P. Fraser, S. Jurek, G. A. Hamil-
ton, K. S. Thorneloe, M. A. McAlexander, D. E. Ingber, Science Trans.
Med. 2012, 4, 159ra147.
[16] A. Jain, R. Barrile, A. van der Meer, A. Mammoto, T. Mammoto, K.
DeCeunyck, O. Aisku, M. A. Otieno, C. S. Louden, G. A. Hamilton, R.
Flaumenhaft, D. E. Ingber, Clin. Pharmacol. Therap. 2018, 103, 332.
[17] J. Li, A. Wen, R. Potla, E. Benshirim, A. Seebarran, M. A. Benz, O.
Henry, R. Prantil-Baun, S. Gilpin, O. Levy, D. E. Ingber, APL Bioeng.
2019, 3, 046103.
[18] K. H. Benam, R. Villenave, C. Lucchesi, A. Varone, C. Hubeau, H.-H.
Lee, S. E. Alves, M. Salmon, T. C. Ferrante, J. Weaver, G. A. Hamilton,
A. Bahinski, D. E. Ingber, Nat. Methods 2016,13, 151.
[19] K. Benam, R. Novak, J. Nawroth, M. Hirano-Kobayashi, T. Ferrante,
Y. Choe, R. Prantil-Baun, A. Bahinski, K. K. Parker, D. E. Ingber, Cell
Syst. 2016, 3, 456.
[20] L. Si, R. Prantil-Baun, K. Benam, H. Bai, M. Rodas, M. Burt, D. E.
Ingber, bioRxiv:685552, 2019, https://doi.org/10.1101/685552.
[21] L. Si, H. Bai, M. Rodas, W. Cao, C. Y. Oh, A. Jiang, R. Moller, D.
Hoagland, K. Oishi, S. Horiuchi, S. Uhl, D. Blanco-Melo, R. A. Al-
brecht, W.-C. Liu, T. Jordan, B. E. Nilsson-Payant, J. Logue, R. Haupt,
M. McGrath, S. Weston, A. Nurani, S. M. Kim, D. Y. Zhu, K. H. Benam,
G. Goyal, S. E. Gilpin, R. Prantil-Baun, R. K. Powers, K. Carlson, M.
Frieman, et al., bioRxiv:04.13.039917, 2020, https://doi.org/10.1101/
2020.04.13.039917.
[22] A. L. Gard, R. Maloney, B. P. Cain, C. R. Miller, R. J. Luu, J. R. Coppeta,
P. Liu, J. P. Wang, H. Azizgolshani, R. F. Fezzie, J. L. Balestrini, B. C.
Isenberg, R. W. Finberg, J. T. Borenstein, bioRxiv:05.23.112797, 2020,
https://doi.org/10.1101/2020.05.23.112797.
[23] H. J. Kim, D. Huh, G. A. Hamilton, D. E. Ingber, Lab Chip 2012, 12,
2165.
[24] H. J. Kim, D. E. Ingber, Integr. Biol. 2013, 5, 1130.
[25] H. J. Kim, H. Li, J. J. Collins, D. E. Ingber, Proc. Natl. Acad. Sci. U. S.
A. 2016, 113, E7.
[26] M. Kasendra, A. Tovaglieri, A. Sontheimer-Phelps, S. Jalili-
Firoozinezhad, A. Bein, A. Chalkiadaki, W. Scholl, C. Zhang, H.
Rickner, C. A. Richmond, H. Li, D. T. Breault, D. E. Ingber, Sci. Rep.
2018, 8, 2871.
[27] A. Sontheimer-Phelps, D. B. Chou, A. Tovaglieri, T. C. Ferrante,
T. Duckworth, C. Fadel, V. Frismantas, S. Jalili-Firoozinezhad, M.
Kasendra, E. Stas, J. C. Weaver, C. A. Richmond, O. Levy, R. Prantil-
Baun, D. T. Breault, D. E. Ingber, Cell. Mol. Gastroenterol. Hepatol.
2020, 9, 507.
[28] M. Kasendra, R. Luc, J. Yin, D. V. Manatakis, G. Kulkarni, C. Lucch-
esi, J. Sliz, A. Apostolou, L. Sunawar, J. Obrigewitch, K.-J. Kang, G. A.
Hamilton, M. Donowitz, K. Karalis, Elife 2020, 9, e50135.
© 2020 The Authors. Published by Wiley-VCH GmbH
www.advancedsciencenews.com
[29] R. Villenave, S. Q. Wales, T. Hamkins-Indik, E. Papafragkou, J. C.
Weaver, T. C. Ferrante, A. Bahinski, C. Elkins, M. Kulka, D. E. Ingber,
PLoS One 2017, 12, e0169412. [47]
[30] S. Jalili-Firoozinezhad, R. Prantil-Baun, A. Jiang, T. Mammoto, J. C.
Weaver, T. C. Ferrante, H. J. Kim, J. M. S. Cabral, O. Levy, D. E. Ingber, [48]
Cell Death Dis. 2018, 9, 223.
[31] A. Grassart, V. Malardé, S. Gobaa, A. Sartori-Rupp, J. Kerns, K. Karalis, [49]
B. Marteyn, P. Sansonetti, N. Sauvonnet, Cell Host Microbe 2019, 26,
435. [50]
[32] S. Jalili-Firoozinezhad, F. S. Gazzaniga, E. L. Calamari, D. M. Cama-
cho, C. W. Fadel, B. Nestor, M. J. Cronce, A. Tovaglieri, O. Levy, K. E.
Gregory, D. T. Breault, J. M. S. Cabral, D. L. Kasper, R. Novak, D. E. [51]
Ingber, Nat. Biomed. Eng. 2019, 3, 520.
[33] T.-E. Park, N. Mustafaoglu, A. Herland, R. Hasselkus, R. Mannix, E. [52]
A. FitzGerald, R. Prantil Baun, A. Watters, O. Henry, M. Benz, H.
Sanchez, H. J. McCrea, L. C. Coumnerova, H. K. Wilson, S. P. Pale-
cek, E. Shusta, D. E. Ingber, Nat. Commun. 2019, 10, 2621.
[34] G. D. Vatine, R. Barrile, M. J. Workman, S. Sances, B. Barriga, M. Rah- [53]
nama, S. Barthakur, M. Kasendra, C. Lucchesi, J. Kerns, N. Wen, W.
R. Spivia, Z. Chen, J. Van Eyk, C. Svendsen, Cell Stem Cell 2019, 24,
995. [54]
[35] G. Morad, C. Carman, E. Hagedorn, J. Perlin, L. Zon, N. Mustafaoglu,
T.-E. Park, D. E. Ingber, C. Daisy, M. Moses, ACS Nano 2019, 13,
13853.
[36] D. B. Chou, V. Frismantas, Y. Milton, R. David, P. Pop-Damkov, D. Fer- [55]
guson, A. MacDonald, O. Vargel-Bolukbasi, C. E. Joyce, L. S. Moreira-
Teixeira, A. Rech, A. Jiang, E. Calamari, S. Jalili Firoozinezhad, B. A.
Furlong, L. R. O’Sullivan, C. F. Ng, Y. Choe, S. Marquez, K. C. Myers, [56]
O. K. Weinberg, R. P. Hasserjian, R. Novak, O. Levy, R. Prantil-Baun,
C. D. Novina, A. Shimamura, L. Ewart, D. E. Ingber, Nat. Biomed. Eng.
2020, 4, 394.
[37] B. Hassell, G. Goyal, E. Lee, O. Levy, C. S. Chen, D. E. Ingber, Cell [57]
Rep. 2017, 21, 508.
[38] A. Marturano-Kruik, M. M. Nava, K. Yeager, A. Chramiec, L. Hao, S.
Robinson, E. Guo, M. T. Raimondi, G. Vunjak-Novakovic, Proc. Natl.
Acad. Sci. U. S. A. 2018, 115, 1256.
[39] S. E. Wheeler, J. T. Borenstein, A. M. Clark, M. R. Ebrahimkhani, I. J. [58]
Fox, L. Griffith, W. Inman, D. Lauffenburger, T. Nguyen, V. C. Pillai, R.
Prantil-Baun, D. B. Stolz, D. Taylor, T. Ulrich, R. Venkataramanan, A.
Wells, C. Young, Stem Cell Res. Ther. 2013, 4, S11.
[40] A. Pavesi, A. T. Tan, S. Koh, A. Chia, M. Colombo, E. Antonecchia, C. [59]
Miccolis, E. Ceccarello, G. Adriani, M. T. Raimondi, R. D. Kamm, A.
Bertoletti, J. CIin. Insight 2017, 2, 89762.
[41] C. Maass, N. B. Sorensen, J. Himmelfarb, E. J. Kelly, C. L. Stokes, M.
Cirit, CPT: Pharmacometrics Syst. Pharmacol. 2019, 8, 316.
[42] E. J. Weber, K. A. Lidberg, L. Wang, T. K. Bammler, J. W. MacDonald,
M. J. Li, M. Redhair, W. M. Atkins, C. Tran, K. M. Hines, J. Herron, L.
Xu, M. B. Monteiro, S. Ramm, V. Vaidya, M. Vaara, T. Vaara, J. Him- [60]
melfarb, E. J. Kelly, JCI Insight 2018, 3, e123673.
[43] J. Wang, C. Wang, N. Xu, Z. F. Liu, D. W. Pang, Z. L. Zhang, Biomate-
rials 2019, 219, 119367.
[44] S. Musah, T. Mammoto, A. Mammoto, T. C. Ferrante, M. Hirano-
Kobayashi, K. Roberts, S. Chung, R. Novak, M. Ingram, T. Fatanat-
Didar, J. C. Weaver, G. M. Church, D. E. Ingber, Nat. Biomed. Eng.
2017, 1, 69.
[45] A. Tovaglieri, A. Sontheimer-Phelps, A. Geirnaert, R. Prantil-Baun, [61]
D. M. Camacho, D. B. Chou, S. Jalili-Firoozinezhad, T. Wouters, M.
Kasendra, M. Super, M. Cartwright, C. A. Richmond, D. T. Breault, C.
Lacroix, D. E. Ingber, Microbiome 2019, 7, 43.
[46] K.-J. Jang, M. A. Otieno, J. Ronxhi, K. Kodella, J. Rubins, D. Petropolis,
G. Kulkarni, D. Simic, H.-K. Lim, W. Lam, M. Singer, E. Barale, B.
Singh, M. Sonee, A. J. Streeter, C. Manthey, L. Ewart, B. Jones, A.
Srivastava, H. Park, J. Nawroth, D. Conegliano, J. Sliz, A. Herland, [62]
Adv. Sci. 2020, 2002030 2002030 (14 of 15)
www.advancedscience.com
S. Haney, D. E. Ingber, G. A. Hamilton, Sci. Trans. Med. 2019, 11,
eaax5516.
X. Li, S. M. George, L. Vernetti, A. H. Gough, D. L. Taylor, Lab Chip
2018, 18, 2614.
N. Tsamandouras, T. Kostrzewski, C. L. Stokes, L. G. Griffith, D. J.
Hughes, M. Cirit, J. Pharmacol. Exp. Ther. 2017, 360, 95.
T. J. Long, P. A. Cosgrove, R. T. Dunn, Drug Metab. Dispos. 2016, 44,
1940.
K. Achberger, C. Probst, J. Haderspeck, S. Bolz, J. Rogal, J. Chuchuy,
M. Nikolova, V. Cora, L. Antkowiak, W. Haq, N. Shen, K. Schenke-
Layland, M. Ueffing, S. Liebau, P. Loskill, Elife 2019, 8, e46188.
K. J. Jang, A. P. Mehr, G. A. Hamilton, L. A. McPartlin, K.-Y. Suh, D. E.
Ingber, Integr. Biol. 2013, 5, 1119.
R. Barrile, A. D. van der Meer, H. Park, J. Fraser, F. Teng, D. Simic, D.
Conegliano, J. Nguyen, T. Almy, D. Weinstock, Y. Wang, C. Louden, K.
Karalis, D. E. Ingber, G. A. Hamilton, M. Otieno, Clin. Pharm. Therap.
2018, 104, 1240.
G. Goyal, B. Bausk, P. Prabhala, L. Xie, D. Curran, J. Long, L. Cohen, O.
Levy, R. Prantil-Baun, D. R. Walt, D. E. Ingber, bioRxiv:806505, 2019,
https://doi.org/10.1101/806505.
B. M. Maoz, A. Herland, E. A. Fitzgerald, T. Grevesse, C. Vidoudez, A.
R. Pacheco, S. P. Sheehy, T.-E. Park, S. Dauth, R. Mannix, N. Budnik,
K. Shores, A. Cho, J. C. Nawroth, D. Segre, B. Budnik, D. E. Ingber, K.
K. Parker, Nat. Biotechnol. 2018, 36, 865.
M. Trapecar, C. Communal, J. Velazquez, C. A. Mauss, Y.-J. Huang, K.
Schneider, C. W. Wright, V. Butty, G. Eng, O. Yilmaz, D. Trumper, L.
G. Griffith, Cell Syst. 2020, 10, 223.
C. W. McAleer, C. J. Long, D. Elbrecht, T. Sasserath, L. R. Bridges,
J. W. Rumsey, C. Martin, M. Schnepper, Y. Wang, F. Schuler, A. B.
Roth, C. Funk, M. L. Schuler, J. J. Hickman, Sci. Transl. Med. 2019, 11,
eaav1386.
L. Vernetti, A. Gough, N. Baetz, S. Blutt, J. R. Broughman, J. A. Brown,
J. Foulke-Abel, N. Hasan, J. In, E. Kelly, O. Kovbasnjuk, J. Repper, N.
Senutovitch, J. Stabb, C. Yeung, N. C. Zachos, M. Donowitz, M. Estes,
J. Himmelfarb, G. Truskey, J. P. Wikswo, D. L. Taylor, Sci. Rep. 2017,
7, 42296.
S. Bauer, C. Wennberg Huldt, K. P. Kanebratt, I. Dureiex, D. Gunne,
S. Andersson, L. Ewart, W. G. Haynes, I. Maschmeyer, A. Winter, C.
Ammala, U. Marx, T. B. Andersson, Sci. Rep. 2017, 7, 14620 [published
correction appears in Sci. Rep. 2018, 8, 1672].
S. Xiao, J. R. Coppeta, H. B. Rogers, B. C. Isenberg, J. Zhu, S. A.
Olalekan, K. E. McKinnon, D. Dokic, A. S. Rashedi, D. J. Haisen-
leder, S. S. Malpani, C. A. Arnold-Murray, K. Chen, M. Jiang, L. Bai,
C. T. Nguyen, J. Zhang, M. M. Laronda, T. J. Hope, K. P. Maniar,
M. E. Pavone, M. J. Avra, E. C. Sefton, S. Getsios, J. E. Burdette,
J. J. Kim, J. T. Borenstein, T. K. Woodruff, Nat. Commun. 2017, 8,
14584.
C. D. Edington, W. L. K. Chen, E. Geishecker, T. Kassis, L. R. Soenksen,
B. M. Bhushan, D. Freake, J. Kirschner, C. Maass, N. Tsamandouras,
J. Valdez, C. D. Cook, T. Parent, S. Snyder, J. Yu, E. Suter, M. Schockley,
J. Velazquez, J. J. Velazquez, L. Stockdale, J. P. Papps, I. Lee, N. Vann,
M. Gamboa, M. E. LaBarge, Z. Zhong, X. Wang, L. A. Boyer, D. A.
Lauffenburger, R. L. Carrier, C. Communal, S. R. Tannenbaum, C. L.
Stokes, D. J. Hughes, G. Rohatgi, D. L. Trumper, M. Cirti, L. G. Griffith,
Sci. Rep. 2018, 8, 4530.
R. Novak, M. Ingram, S. Marquez, D. Das, A. Delahanty, A. Herland,
B. M. Maoz, S. S. F. Jeanty, M. R. Somayaji, M. Burt, E. Calamari,
A. Chalkiadaki, A. Cho, Y. Choe, D. B. Chou, M. Cronce, S. Dauth, T.
Divic, J. Fernandez-Alcon, T. Ferrante, J. Ferrier, E. A. FitzGerald, R.
Fleming, S. Jalili-Firoozinezhad, T. Grevesse, J. A. Goss, T. Hamkins-
Indik, O. Henry, C. Hinojosa, T. Huffstater, et al., Nat. Biomed. Eng.
2020, 4, 407.
H. E. Abaci, M. Shuler, Integr. Biol. 2015, 7, 383.
© 2020 The Authors. Published by Wiley-VCH GmbH
www.advancedsciencenews.com www.advancedscience.com

[63] M. B. Esch, H. Ueno, D. R. Applegate, M. L. Shuler, Lab Chip 2016,
16, 2719.
[64] J. R. Coppeta, M. J. Mescher, B. C. Isenberg, A. J. Spencer, E. S. Kim, A.
R. Lever, T. J. Mulhern, R. Prantil-Baun, J. C. Comolli, J. T. Borenstein,
Lab Chip 2016, 17, 134.
[65] I. Wagner, E.-M. Materne, S. Brincker, U. Süssbier, C. Frädrich, M.
Busek, F. Sonntag, D. A. Sakharov, E. V. Trushkin, A. G. Tonevitsky, R.
Lauster, U. Marx, Lab Chip 2013, 13, 3538.
[66] C. L. Stokes, M. Cirit, D. Lauffenburger, CPT: Pharmacometrics Syst.
Pharmacol. 2015, 4, 559.
[67] D. Bovard, A. Sandoz, K. Luettich, S. Frentzel, A. Iskandar, D.
Marescotti, K. Trivedi, E. Guedj, Q. Dutertre, M. C. Peitsch, J. Hoeng,
Lab Chip 2018, 18, 3814.
[68] C. Oleaga, B. Bernabini, A. S. Smith, B. Srinivasan, M. Jackson, W.
McLamb, V. Platt, R. Bridges, Y. Cai, N. Santhanam, B. Berry, S. Najjar,
N. Akanda, X. Guo, C. Martin, G. Ekman, M. B. Esch, J. Langer, G.
Ouedraogo, J. Cotovio, L. Breton, M. L. Shuler, J. J. Hickman, Sci.
Rep. 2016, 6, 20030.
[69] I. Maschmeyer, A. K. Lorenz, K. Schimek, T. Hasenberg, A. P. Ramme,
J. Hübner, M. Lindner, C. Drewell, S. Bauer, A. Thomas, N. S. Sambo,
F. Sonntag, R. Lauster, U. Marx, Lab Chip 2015, 15, 2688.
[70] A. Herland, B. M. Maoz, D. Debarun, M. R. Somayaji, R. Prantil-
Baun, R. Novak, M. Cronce, T. Huffstater, S. S. F. Jeanty, M. In-
gram, A. Chalkiadaki, D. B. Chou, S. Marquez, A. Delahanty, S. Jalili-
Firoozinezhad, Y. Milton, A. Sontheimer-Phelps, B. Swenor, O. Levy,
K. K. Parker, A. Przekwas, D. E. Ingber, Nat. Biomed. Eng. 2020, 4,
421.
[71] S. Sances, R. Ho, G. Vatine, D. West, A. Laperle, A. Meyer, M.
Godoy, P. S. Kay, B. Mandefro, S. Hatata, C. Hinojosa, N. Wen, D.
Sareen, G. A. Hamilton, C. N. Svendsen, Stem Cell Rep. 2018, 10,
1222.
[72] S. Peel, A. M. Corrigan, B. Ehrhardt, K.-J. Jang, P. Caetano-Pino, M.
Boeckeler, J. E. Rubins, K. Kodella, D. B. Petropolis, J. Ronxhi, G.
Kulkarni, A. J. Foster, D. Williams, G. A. Hamilton, L. Ewart, Lab Chip
2019, 19, 410.
[73] S. B. Kim, K.-I. Koo, H. Bae, M. R. Dokmeci, G. A. Hamilton, A. Bahin-
ski, S. M. Kim, D. E. Ingber, A. Khademhosseini, Lab Chip 2012, 12,
3976.
[74] M. Odijk, A. D. van der Meer, D. Levner, H. J. Kim, M. W. van der
Helm, L. I. Segerink, J. P. Frimat, G. A. Hamilton, D. E. Ingber, A. van
den Berg, Lab Chip 2015, 15, 745.
[75] S. A. Mousavi, F. De Ferrari, Y. S. Zhang, M. Nabavinia, N. B. Mo-
hammad, J. Ryan, A. Pourmand, E. Laukaitis, R. B. Sadeghian, A.
Nadhman, S. R. Shin, A. S. Nezhad, A. Khademhosseini, M. R. Dok-
meci, Biomicrofluidics 2016, 10, 044111.
[76] O. Y. F. Henry, R. Villenave, R. D. E. Ingber, Lab Chip 2017, 17, 2264.
[77] B. M. Moaz, A. Herland, O. Y. F. Henry, W. Leineweber, M. Yadid, R.
Mannix, V. J. Kujala, E. A. Fitzgerald, K. K. Parker, D. E. Ingber, Lab
Chip 2017, 17, 2294.
[78] D. Sheyn, D. Cohn-Yakubovich, S. Ben-David, S. De Mel, V. Chan,
C. Hinojosa, N. Wen, G. A. Hamilton, D. Gazit, Z. Gazit, Microfluid.
Nanofluid. 2019, 23, 99.
[79] Y. S. Zhang, J. Aleman, S. R. Shin, T. Kilic, D. Kim, S. A. M. Shaegh,
S. Massa, R. Riahi, S. Chae, N. Hu, H. Avci, W. Zhang, A. Silvestri,
A. S. Nezhad, A. Manbohi, F. D. Ferrari, A. Polini, G. Calzone, N.
Shaikh, P. Alerasool, E. Budina, J. Kang, N. Bhise, J. Ribas, A. Pour-
mand, A. Skardal, T. Shupe, C. E. Bishop, M. R. Dokmeci, A. Atala, A.
Khademhosseini, Proc. Natl. Acad. Sci. U. S. A. 2017, 114, E2293.

Donald Ingber is the founding director of the Wyss Institute for Biologically Inspired Engineering at
Harvard University, Judah Folkman Professor of Vascular Biology at Harvard Medical School and
Boston Children’s Hospital, and professor of bioengineering at the Harvard John A. Paulson School
of Engineering and Applied Sciences. He received his B.A., M.A., M.Phil., M.D., and Ph.D. from Yale
University. He has made significant contributions to many disciplines and helped to pioneer the devel-
opment of microfluidic Organ Chips.

Adv. Sci. 2020, 2002030 2002030 (15 of 15) © 2020 The Authors. Published by Wiley-VCH GmbH