Organ-on-Chips Revolutionize Drug Safety Testing
Whitepaper
Last Updated: March 21, 2024
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Published: March 15, 2024
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Both animal and in vitro 2D cell culture models used during preclinical trials cannot faithfully replicate the physiological conditions of human in vivo cellular environments. As a result, over 90% of new compounds fall short during clinical trials.
Organ-on-chip (OOC) models have emerged as a way to replicate the complex microenvironment of human organs in vitro, allowing researchers to test drugs and study diverse physiological processes.
This whitepaper explores the latest advancements in OOC platforms that can help to efficiently simulate drug toxicity responses.
Download this whitepaper to discover:
- Liver- and kidney-on-chip models for simulating toxicity responses
- High-content imaging to visualize cell interactions, structures and responses
- How OOC models can reduce development costs and improve success rates
LITERATURE REVIEW
Organ-on-chips: increased complexity
for higher physiological relevance in
pharmaceutical safety testing.
With as many as 90% of new drug compounds falling short in
clinical trials, the journey from a promising drug candidate to
a marketable medication is fraught with challenges and
uncertainties. This high attrition rate is not only costly for
drug developers but also hinders access to better and
potentially life-saving treatments for patients. Even with
innovative tools such as artificial intelligence, machine
learning, 3D cell cultures, and omics-based technologies,
the prevalence of drug failures continues to cast a shadow
on the promise of breakthrough therapies.
One of the major reasons for the failure of many new
medicines is the inability to accurately predict drug
responses in vivo. Although animal models are widely used
for preclinical and toxicity testing, the results obtained often
inaccurately reflect human physiology due to interspecies
differences. Animal studies are also costly and
time-consuming and have raised ethical and animal welfare
concerns. Despite considerable advances in understanding
the pathophysiological differences and similarities between
patients and preclinical animal models, there are still
instances of over- or underestimating cellular behaviors and
drug responses in preclinical phases, especially when
addressing efficacy and toxicity. This discrepancy has
resulted in drugs that successfully pass through preclinical
development only to fail in later clinical stages.
For research use only. Not for use in diagnostic procedures.
A notable step in addressing the reliance on animal testing
was the U.S. Congress’s passage of the FDA Modernization
Act 2.0 in 2023. This authorizes the use of certain
alternatives to animal testing for drug and biological product
applications, such as cell-based assays and computer
models, to investigate the safety and effectiveness of a drug.
The legislation also removes a requirement to use animal
studies as part of the process to obtain a license for a
biological product that is biosimilar or interchangeable with
another biological product.
While these efforts are a step forward from an ethical
standpoint, it also presumes that alternative research
models are available for preclinical testing. One easily
accessible alternative involves in vitro 2D cell culture
models using primary or immortalized cell lines. The
challenge with these models is that they cannot faithfully
replicate the physiological conditions of in vivo cellular
environments, such as the spatial organization,
dimensionality of the extracellular matrix (ECM), and critical
cell-cell and cell-ECM interactions. There is therefore a need
for the development of accurate and reliable in vitro models
with acceptable biological relevance to accelerate the
development of new drugs, while also mitigating the risk of
costly failures. These models are required to bridge the gap
between traditional testing methods and in vivo biology,
ensuring a seamless transition from promising candidates to
efficacious medications for patients.
Organ-on-chips: increased complexity for higher physiological relevance in pharmaceutical safety testing.
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Modeling toxicity with organ-on-chip
platforms
Organ-on-chip (OOC) models have emerged as a promising
alternative, potentially overcoming some of the limitations of
animal experiments. An OOC is a microfluidic device
containing hollow channels lined with living cells, which are
often derived from human tissues or stem cells, to form
miniature organ-like structures. The cells can interact with
each other and the microenvironment to mimic functional
units of specific organs. The primary goal of OOC technology
is to replicate the complex microenvironment of human
organs in vitro, allowing researchers to study diverse
physiological processes, test drugs, and gain insights into
human biology. One of the attractive features of OOC devices
is their ability to reproduce mechanical forces and physical
characteristics unique to each organ. For example,
lung-on-chip devices may simulate breathing motions, while
heart-on-chip devices may replicate the heart’s rhythmic
contractions.
Because the failure of many drug candidates is often attributed
to their toxicity in humans, with the liver and kidneys being
common sites of adverse effects, leveraging OOCs in drug
safety assessments allows researchers to closely mimic the
physiological conditions of these vital organs in vitro. This
approach can provide valuable insights into potential toxicities
and help to identify compounds with improved safety profiles
early in the drug development process. In this article, we
explore some advancements of liver and kidney-on-chip
models, illustrating how these platforms empower researchers
to simulate toxicity responses.
Liver-on-chip models for evaluating
hepatotoxicity
Drug-induced liver injury (DILI) is one of the primary toxicities
that cause drugs to be withdrawn from the market and a
major safety concern in pharmaceutical development.
Increasing numbers of liver-on-chip platforms are being
developed to model drug metabolism, drug-drug interactions,
and hepatotoxicity. These devices typically contain human
liver cells, such as hepatocytes, and are designed to mimic
the structural and functional complexity of the liver tissue.
While liver-on-chip technology holds promise for advancing
drug development, the models need to replicate the liver’s
histological structures and functions and accurately
distinguish between toxic and non-toxic drugs.
In a study conducted by Ewart et al., the team investigated
the efficacy of a liver-on-chip platform (Liver-Chip) in
recapitulating the human liver structure and predicting DILI.1
Successful mimicry of the structural and cellular organization
of the human liver increases the likelihood that the device will
also replicate its functional capabilities.
Figure 1: Recapitulation of human liver structure in the Liver-Chip.
Representative phase contrast microscopic images (scale bar
represents 10 µm) of hepatocytes in the upper channel of LiverChip (a) and non-parenchymal cells in the lower vascular channel
(the regular array of circles are the pores in the membrane) (b).
Representative immunofluorescence microscopic images showing
the phalloidin stained actin cytoskeleton (green) and ATPBcontaining mitochondria (magenta) (c) and MRP2-containing bile
canaliculi (red) (d). CD31-stained liver sinusoidal endothelial cells
(green) and desmin CD68+ containing stellate cells (magenta) (e),
and Kupffer cells (green) co-localized with desmin-containing
stellate cells (magenta) (f). All images in c–f show DAPI-stained
nuclei (blue) and the scale bar represents 100 µm with the inset at
5 times higher magnification. Image source: Ewart L, Apostolou A,
Briggs SA, Carman CV, Chaff JT, Heng AR, et al. Performance
assessment and economic analysis of a human Liver-Chip for
predictive toxicology. Communications Medicine. 2022;2(1). Image
licensed under Creative Commons License 4.0.
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Organ-on-chips: increased complexity for higher physiological relevance in pharmaceutical safety testing.
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Leveraging the Opera Phenix® high-content screening system
the researchers confirmed the presence of distinct
hepatocytes and liver sinusoidal endothelial cells (LSECs)
within the Liver-Chip (Figures 1a-b). They also observed
liver-specific structures such as bile canaliculi, mitochondrial
markers in hepatocytes, and specific cellular markers for
LSECs, Kupffer cells, and stellate cells (Figures 1c-f). Once
they had confirmed the model’s ability to sustain hepatocyte
functionality, the team investigated the ability of the
liver-on-chip platform to predict DILI across a blinded set of
27 drugs, each with a known hepatotoxic or non-toxic
behavior. The Liver-Chip correctly identified 87% of drugs
causing DILI, outperforming both 3D liver and animal models.
Additionally, no drugs were falsely marked as toxic,
highlighting the potential of liver-on-chip models to identify
hepatotoxic compounds and revolutionize drug development
processes. Beyond the scientific validation, the researchers
also noted the profound economic implications of adopting
such technologies due to increased R&D productivity. This
suggests that liver-on-chip models could improve the
efficiency of drug development and also offer substantial
economic benefits.
Kidney-on-chip models for evaluating
nephrotoxicity
In addition to DILI, drug-induced nephrotoxicity remains a
significant concern in pharmaceutical development and has
led to the withdrawal of various medications from the market
due to unforeseen renal complications. Early detection of
potential nephrotoxic effects is therefore critical not only to
avert costly late-stage setbacks but also to aid the
development of safer therapeutic options for patients.
Central to effective nephrotoxicity testing is the ability to
develop physiologically relevant models of the proximal
tubule (PT). This site of the nephron is crucial for drug
clearance and is one of the primary sites susceptible to
drug-induced renal damage. Although various animal and in
vitro models of the kidney exist, they often fail to capture the
intricate functions and responses of the PT to drug exposures.
The emergence of advanced kidney-on-chip platforms
therefore offers a promising avenue for more physiologically
relevant nephrotoxicity testing. Yet, replicating the PT’s
structure and function poses specific challenges, especially
given that reabsorption within the PT occurs across opposing
monolayers of epithelium and endothelium separated by a
basement membrane.
A study conducted by Vedula and colleagues involved the
development of a kidney-on-chip model designed to address
these complexities.2
The model integrated human renal PT
epithelial cells (hRPTEC) and human microvascular endothelial
cells (hMVEC) within a co-culture setting. The setup aimed to
facilitate the formation of PT tissue structures that closely
resemble the in vivo environment while also allowing for
real-time quantification of renal reabsorptive functions. After
confirming that the co-culture architecture successfully
mimicked the physiological structure of a renal PT, the
researchers used a fluorescent glucose analog (2-NBDG)
to monitor reabsorption in real-time in the presence of the
Na+
/K+
-ATPase inhibitor, ouabain. The dynamic responses in
glucose reabsorption the team observed validated the
model’s ability to reproduce a crucial function of the PT. The
researchers note that, unlike previous models, their
microfluidic PT model not only mimicked the physiological
architecture but also provided direct evidence of active
reabsorptive functionalities across epithelial and endothelial
cell monolayers.
Building on this work, another research effort led by Erin
Shaughnessey evaluated the use of a kidney-on-chip model to
detect drug-induced nephrotoxicity in primary PT cells,
leveraging the high-throughput PREDICT96 microfluidic
system.3
The PREDICT96 platform enables parallel culture of
96 individual co-culture devices and control of fluid flow
within the footprint of a conventional multi-well culture plate
(Figure 2). Through this platform, the researchers were able to
evaluate the effectiveness of transepithelial electrical
resistance (TEER) sensing in identifying cisplatin-induced
toxicity within human primary PT models. Their investigation
spanned various conditions, including mono- and co-culture
settings and differing levels of fluid shear stress (FSS).
While both models responded to cisplatin-induced toxicity,
only the hRPTEC-hMVEC co-culture model showed TEER
changes correlating with cytotoxicity and tight junction
alterations. Importantly, the TEER measurements within the
co-culture model indicated the emergence of cisplatininduced toxicity earlier than observable cell death,
showcasing its potential as an early, non-invasive indicator
of drug-induced nephrotoxicity in a high-throughput
screening setting. Such findings position the PREDICT96
platform as a pivotal tool in high-throughput nephrotoxicity
screening endeavors and underscore the potential of TEER
as an early and non-invasive biomarker for detecting
drug-induced nephrotoxicity.
Organ-on-chips: increased complexity for higher physiological relevance in pharmaceutical safety testing.
www.revvity.com 4
Future outlook
Advancements in OOC models have enabled microfluidic
platforms to emulate the microenvironments of various human
organs with remarkable fidelity. Particularly, liver- and
kidney-on-chip platforms are enhancing the predictive
accuracy of drug-induced toxicities such as hepatotoxicity and
nephrotoxicity, respectively. The microfluidic systems in OOC
devices enable the continuous perfusion of nutrients, oxygen,
and other essential factors to the cells. This perfusion system
helps maintain cell viability and function over extended time
periods, potentially allowing for studies involving longer-term
processes, such as chronic drug exposure, or disease
progression. By utilizing high-content imaging, OOCs can
provide detailed visualizations of cell interactions, structures,
and responses within the microfluidic devices, enhancing the
accuracy of drug toxicity assessments and facilitating the
development of safer and more effective medicines.
As the scientific community becomes more aware of the
advantages of OOCs over traditional models, their adoption
will likely continue to increase. This will be driven by the
pressing need to reduce drug development costs, improve
success rates, and address ethical concerns related to
animal testing. With growing interest from pharmaceutical
companies, we can expect more significant investments in
OOC technologies leading to the development of
standardized OOC platforms, making them more accessible
and widely adopted across the industry.
Figure 2: The PREDICT96 platform with integrated TEER sensing supports proximal tubule-microvascular co-culture in vivo-like features. (a) The
PREDICT96 culture plate has 96 microfluidic devices (top left). Cross-sectional rendering of PREDICT96 Integrated TEER system (bottom) highlights
the four-point TEER measurement unit (illustration, top right) in which the stainless steel pump tubes double as electrodes. (b) A cross-section
schematic of the co-culture kidney model in the bilayer microfluidic device with hRPTEC on the bottom of the membrane and hMVEC on top of
the membrane. Fluid flow is controlled separately in the top and bottom channels. (c) A confocal tile scan of a PREDICT96 device shows confluent
cell layers under high FSS (0.70 dyn/cm2
) on day 7 (Calcein AM, green). (d) An orthogonal view of a confocal z-stack shows hRPTEC and hMVEC on
either side of the device membrane (dashed line) with hRPTEC expressing apical ZO-1 (green) and hMVEC expressing endothelial marker von
Willebrand factor (vWF, red). (e, top) A maximum intensity projection of a z-stack of hRPTEC on the bottom side of the membrane shows
continuous tight junctions (ZO- 1, green) and abundant primary cilia (acetylated tubulin, red). (e, bottom) An orthogonal view demonstrates hRPTEC
apical expression of ZO-1 and primary cilia. (f) A confocal z-slice of hMVEC on the top side of the membrane shows a characteristic punctate
expression of vWF (red) and tight junctions (green). Image source: Shaughnessey EM, Kann SH, Azizgolshani H, Black LD, Charest JL, Vedula EM.
Evaluation of Rapid Transepithelial Electrical Resistance (teer) measurement as a metric of kidney toxicity in a high-throughput microfluidic culture
system. Scientific Reports. 2022;12(1). Image licensed under Creative Commons License 4.0.
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Organ-on-chips: increased complexity for higher physiological relevance in pharmaceutical safety testing.
References
1. Ewart L, Apostolou A, Briggs SA, Carman CV, Chaff
JT, Heng AR, et al. Performance assessment and
economic analysis of a human Liver-Chip for predictive
toxicology. Communications Medicine. 2022;2(1).
doi:10.1038/s43856-022-00209-1
2. Vedula EM, Alonso JL, Arnaout MA, Charest JL.
A microfluidic renal proximal tubule with active
reabsorptive function. PLOS ONE. 2017;12(10).
doi:10.1371/journal.pone.0184330
3. Shaughnessey EM, Kann SH, Azizgolshani H, Black
LD, Charest JL, Vedula EM. Evaluation of Rapid
Transepithelial Electrical Resistance (teer) measurement
as a metric of kidney toxicity in a high-throughput
microfluidic culture system. Scientific Reports. 2022;12(1).
doi:10.1038/s41598-022-16590-9
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