Organ-on-a-Chip Devices as a Tool for

Drug Testing
Meghan Tighe

Abstract
The drug discovery and development process is long, ineffective, and expensive, delaying
accessibility of effective drugs to patients who need them (Wikswo, Esch, Maschmeyer).
Organ-on-a-chip (OoC) devices have been proposed as an additional component of the
preclinical testing phase to improve this process. OoC devices are microfluidic devices in
which cells can be cultured to represent one or more functions of an organ. They are
largely successful models due to their relevant physics, incorporation of crosstalk between
cell types, ability to accurately represent barriers, and potential for individualized treatment
testing (Esch). Due to the ease of microfabrication and soft lithography, use of OoC devices
is financially advantageous as a method for preclinical screening of drugs compared to
current standard methods (Huh 2013). The largest challenges for OoC devices include
determination and implementation of appropriate scaling to represent organ function and
overcoming limitations in analysis methods due to small volume. OoC devices have great
potential and research on these challenges should be prioritized in order to advance their
capabilities and promote them as a tool for the drug development process.

Introduction
The Drug Development Process
Drug discovery and development is a long process that leads up to the U.S. Food & Drug
Administration (FDA) approval process (Figure 1). On average, the discovery, development,
and approval process for one drug takes twelve years and costs $2.6 billion (Somerville,
Phrma). After drugs have been discovered and selected for testing, a thorough preclinical
testing phase is required before human clinical trials can begin. The purpose of preclinical
studies is to investigate toxicity and dosing so that feasibility of clinical trials can be
assessed and the parameters of those clinical trials can be determined (FDA). The focus is
investigation of the absorption, distribution, metabolism, and elimination of drugs that are

introduced to the human body (Esch). This phase of testing begins with mathematical
modeling and is followed by in vitro testing and animal testing.

Figure 1: Current Standard Drug Development Process. The drug development process
includes drug discovery, preclinical testing, clinical trials, and submission to and review by
the FDA. This process generally takes over a decade and results in the approval of one
drug. Figure reprinted from Somerville.
Mathematical models can be extremely powerful tools when correctly informed.
Pharmacokinetic models are simple, compartment-based models that aim to show the
overall distribution of a drug throughout the body over time without focusing on the
mechanisms at play. Physiologically based pharmacokinetic models are more complex,
typically have more compartments, and better incorporate biochemical effects (Esch).
Another common method, pharmacodynamic modeling, goes one step farther and
investigates the effects that a drug has on the body based on its concentration (Abaci).
These models are used in combination to mathematically model what happens when a
drug is introduced to the human body. They can yield useful representations of specific
parts of the body. Use of mathematical models includes a lot of guesswork and the biggest
roadblock with mathematical modelling is finding accurate parameters (Wikswo & Block
2013).
In vitro models use cells and thus do not require parameters to be quantified in the
same way that mathematical models do. The use of living cells takes out a lot of the
guesswork that is necessary in mathematical modelling. In vitro models still have major
limitations however. Typically, immortalized cell lines are used in these experiments due to
their availability. Unfortunately, immortalized cell lines are frequently not accurate
representations of normal human cells (Esch). The typical in vitro models used for
pharmacological research are two-dimensional, meaning that they incorporate neither the
geometry in which cells exist in vivo nor extracellular matrix (ECM) components (Helm).
Two-dimensional models struggle to incorporate physical phenomena such as shear stress,

which can greatly influence cell behavior and study results. The lack of ECM components
further limits in vitro because cellular behavior is greatly influenced by its environment in
the extracellular matrix (Esch).
Another core component of preclinical drug development is animal testing. Animal
models are able to capture many of the complex interactions and less understood
mechanisms that cannot be recreated in vitro. Inclusion of these complexities does not
always lead to predictive results though (Huh 2010). In fact, more than 80% of drugs that
prove successful in animal models fail in human clinical trials (Helm). It is also thought that
animal trials could falsely rule out drugs that would be successful in clinical trials (Esch).
The use of animals for preclinical testing is costly and labor intensive (Helm). There are also
major ethical concerns regarding the use of animals to test products for humans, especially
given the ineffectiveness of the testing (Esch). As experimental subjects, these animals take
drugs that are still considered too risky for human trials. Additionally, in many cases, the
animals being testing upon are given disease states. Although animal testing is considered
the norm, it is considered unethical by many people (Singer).
Current methods of testing in the preclinical phase of the drug development
process are costly and inefficient. The lengthy development process delays availability of
drugs to patients in need. Better models could make drug development not only faster and
less costly, but also more accurate (Esch).

Organ-on-a-Chip
The use of organ-on-a-chip (OoC) devices has been introduced as a possible
addition to the preclinical phase of drug development testing (Figure 2). These models are
microfluidic devices that incorporate chambers and microchannels, on one or multiple
layers, in which human cells can be cultured (Figure 3). The devices are made to represent
parts of the human body by providing an environment that is more relevant than that
provided in traditional in vitro modeling. Variations of these devices have been made under
names such as micro cell culture analog devices, multicompartmental bioreacters, and
human-on-a-chip devices (Esch, Guzzardi, Abaci).

Figure 2: Proposed Steps of a Drug Development Process Utilizing Organ-on-a-Chip

Devices. Organ-on-a-chip devices (OOC) or human-on-a-chip devices (HOC) could be
utilized throughout the drug development process, simultaneously taking discoveries from
and informing mathematical models, animal models, and clinical trials. Use of these
devices late in the drug development process could include testing for development of
individualized treatment. Figure reprinted from Abaci.
Drugs are often taken because there is already a problem in the body. In cases of
well understood conditions, organ-on-a-chip devices can be made to model diseased as
well as healthy organs, either by using diseased cells or by adjusting the geometry of the
microfluidic device to reflect altered function (Wikswo & Block 2013, Wlodkowic).

Figure 3: Simple Organ-on-a-Chip Device. A) The layered components of a OoC device. B)

Photo showing size of final device. Figure adapted from Bhatia.

Use of Microfluidic Devices

The design of each organ-on-a-chip device is dependent on the organ that is being
modeled. Different systems can be modeled through design of the geometry of the device
as well as types, combinations, and locations of cells cultured in the device. Once designed,
OoC devices are most commonly manufactured through microfabrication and
photolithography.

Microfabrication
The photolithographic fabrication process usually utilized in the construction of
microfluidic devices allows for construction of features that are at the same scale that cells
can sense and respond to tissue. Typically, OoC devices are made with
polydimethylsiloxane (PDMS) due to the ease with which it can be cast and bonded to
glass. Although PDMS is the standard, silicon and glass were originally more commonly
used and are still viable options; under certain circumstances these materials offer
important advantages. For example, the mechanical properties of PDMS make it prone to
collapsing in wide channels for instances in which excessively wide channels are necessary,
other materials may be preferable (Iliescu).

Figure 4: PDMS Microfluidic Device Fabrication Process. a) Photolithography. A silicon

wafer is spin-coated with photoresist. Photomask is put in place and ultraviolet light is
applied to make features. After a wash, replica molding yields a PDMS stamp with
complementary features. b) Soft Lithography. Replica molding can take place many times
to make many PDMS stamps. Further preparation includes hollowing of the inlets and
outlets and bonding the PDMS stamp to a glass slide. Figure reprinted from Bhatia.

The first half of the manufacturing process requires photolithography (see Figure
4a). First, a clean silicon wafer is spin-coated with a thin layer of photoresist. Next, a
photomask is layered above it. This photomask, usually designed through computer aided
design, has opaque patterns that protect some areas from the ultraviolet rays that are
applied next. After being treated with ultraviolet light, the photomask is removed. The
photoresist that was not protected by the photomask, and thus received treatment by the
ultraviolet light, is now bonded to the silicon wafer. Excess photoresist is removed and
features corresponding to the pattern on the photomask can be seen engraved into the
photoresist. This product of this process, a master wafer, can be used indefinitely to make
PDMS stamps (Figure 4b).
A PDMS stamp is the flexible PDMS part of a microfluidic device, which will later be
bound to a glass slide. It is molded through replica molding. In this process PDMS, mixed
with a curing agent, is poured onto the wafer. The PDMS is cured in an oven and can then
be peeled from the master wafer, resulting in a PDMS stamp. Any inlet or outlet ports are
stamped at this point. Next, the PDMS stamp is sealed permanently to a glass slide with the
features contacting the slide, creating channels between the glass and the PDMS, as shown
in Figure 5 (McDonald).

Figure 5: PDMS Microfluidic Device. A PDMS stamp with features is bonded to a glass
slide to create hollow microchannels.
Master wafers can be ordered from suppliers and used indefinitely. Unlike
photolithography which requires a cleanroom, soft lithography can be done in a standard
environment. This makes construction of microfluidic devices accessible and inexpensive
(McDonald, Iliescu).

System Set Up
Devices that incorporate a matrix component require additional steps before cells can be
introduced. Devices looking at barriers may only require ECM components coating the
sides of the channels. In their model of the alveolar-capillary barrier, Huh et al. coated their
devices microchannels with an ECM coating solution. After rinsing, they were left with

hollow channels coated in fibronectin and collagen (Huh 2013). Alternatively, some devices
have channels or chambers filled with matrix components. Nguyen et al. used two parallel
needles to insert collagen I into a channel in their device. After letting the collagen set, they
removed the needles, leaving two hollow channels completely surrounded by collagen
(Nguyen). Variations of these two techniques allow scientists to incorporate ECM
components to model the interactions relevant to the function being studied.
Cells can now be introduced from one or more inlets. The typical method of filling
devices uses bent needles which are attached to tubing. Media is sent through tubes by a
syringe pump (Figure 6). Multistep techniques exist and can be modified to seed cells into
specific areas of the devices or to create gradients (Huh 2013).

Figure 6: Fully Constructed OoC System. Bent needles are connected to tubes and
inserted into inlet and outlet ports. Tubes are powered by a syringe pump. Figure adapted
from Huh 2013, Yin.
Once cells and media have been introduced, the experiment can run its course.
Most OoC experiments last between 24 and 72 hours with very few lasting over a week
(Maschmeyer). Methods of measurement and analysis will be discussed later in this text.

Features
There are many features of organ-on-a-chip devices that could allow them to be successful
models for use during the preclinical phase of drug development. The devices allow for
relevant physics and thus relevant physical phenomena. They also allow for co-culturing in
which cross talk between different cell types can be incorporated. Organ-on-chip devices
serve as a possible mechanism of testing for individualized treatment. Finally, these
systems provide a useful model of many barriers in the body.

Physics
Organ-on-a-chip devices are microscale models that are designed to represent the
geometry of the environment in which cells exist in the body. This relevant size and

geometry leads to relevant physics. Important physical phenomena in microfluidic devices

include laminar flow and shear stress, diffusion, surface area to volume ratio, and surface
tension (Helm, Vogel, Guo, Beebe). OoC devices are designed so that these physical
properties represent those of the system being modeled.
Jang et al. conducted a study in which human kidney endothelial cells were cultured
in a microfluidic device. The design of the device allowed for flow of media which
represented the tubular flow through the human kidney proximal tubule in which the cells
are located in vivo. Incorporation of flow, and thus shear stress, resulted in physiologically
relevant expression of cilia and other appropriate cell activity. The activity evaluated in this
study showed closer resemblance to in vivo studies than those seen in previous in vitro
studies (Jang).
Organ-on-a-chip devices can incorporate forces that cells experience in the body.
Huh et al. designed a lung-on-a-chip device that allowed them to study the effect of the
cyclic mechanical strain that cells undergo in the alveoli due to breathing. A vacuum was
used to apply a physiological strain, made possible by the flexibility of the PDMS walls
(Figure 7a). Results showed that cyclical strain increases alveolar epithelial cells production
of proinflammatory cytokines. This is demonstrated in Figure 7b by an increased
production of reactive oxygen species (ROS) measured through microfluorimetry (Huh
2010).

Figure 7: Reactive Oxygen Species Production by Alveolar Epithelial Cells. Use

of an organ-on-a-chip device incorporating mechanical strain (A) allowed Huh et al. to
discover increased production of proinflammatory cytokines as measured through
microfluorimetry (B). Figure adapted from Huh 2010.
In another study, Huh et al. investigated the injury of human airway epithelial cells
that occurs due to liquid plugs that present due to surfactant dysfunction in conditions
such as asthma and pneumonia. Previous studies in this area had already modeled
presentation of the liquid plugs as semi-infinite bubbles, both mathematically and in in vitro
models. These studies, performed experimentally with parallel plate apparatuses, had not

been able to model the rupture of these bubbles. Due to their small, biologically relevant
geometry, OoC devices allowed Huh et al. to expand the current models by incorporating
this final activity (Huh 2007).

Cross talk
Organ-on-a-chip devices allow for culturing of multiple cell types in separate but connected
compartments, something that is not done with standard pharmacological in vitro testing
equipment. This allows for interactions that cant be represented through use of only one
cell type nor through uniform co-culture of multiple cell types (Esch, Guzzardi).
Incorporation of this cell to cell communication is critical to understanding and
representing in vivo cellular behavior (Guo). The small size of OoC devices allows for more
precise control over interactions between cells.
Cross talk is extremely important in studies of immune system interactions.
Businaro et al. utilized PDMS microfluidic devices to co-culture B16-F10 melanoma cells
with murine immune cells isolated from the spleens of wild type (WT) or interferon
regulatory factor-8 (IRF-8) deficient (KO) mice to investigate the role of IRF-8 in immune cell
crosstalk with cancer cells. The two types of cells were cultured in separate chambers
connected with microchannels. Businaro investigated movement of the immune cells
through time-lapse fluorescence microscopy for a period of 48 hours. As shown in Figure 8,
WT immune cells showed clear migration towards melanoma cells, while the migration of
IRF-8 KO cells showed little correlation to the direction of the cancer cells. The use of
microfluidic devices in this study allowed for easy facilitation of crosstalk between different
cell types and a straightforward method of tracking individual cell movements (Businaro).

Figure 8: Immune Cell Migration Due to Crosstalk. This figure shows the migration of
both wild type and interferon regulatory factor-8 deficient murine immune cells towards
melanoma cells. For each cell: The origin is shown as a black circle, the path of migration is

shown in purple, and the final position is shown as a red circle. Figure adapted from
Businaro.
Guzzardi et al. used an OoC device to investigate crosstalk between hepatocytes and
endothelial cells. They co-cultured HepG2 hepatic cells with human umbilical vein
endothelial cells, both in a static well culture and in a microfluidic device
(multicompartmental bioreacter, MCB). Crosstalk was analyzed through measurements of
glucose consumption and secretion of albumin, urea, and nitric oxide. These metabolites
were measured in extracted media with commercial kits. Use of an OoC device caused
significant changes in crosstalk as demonstrated by the criteria chosen. Figure 9 shows the
comparisons for urea secretion: use of a OoC device increased representation of crosstalk
such that urea secretion increased more than tenfold. This device, as well as OoC devices in
general, represents a more accurate model by better incorporating crosstalk between the
co-cultured cells and offers possibilities for study of lesser known effects of crosstalk
(Guzzardi).

Figure 9: Crosstalk between hepatocytes and endothelial cells. Figure compares

urea secretion of: hepatocytes alone and hepatocytes co-cultured with human umbilical
vein endothelial cells; cultured in a static culture and in a OoC device. Figure adapted from
Guzzardi.

Individualized Treatment
The technology in question also offers potential for individualized treatment investigation.
Tuning the parameters of a mathematical model to those of an individual is impossible
without testing and measuring the individuals response to drugs. Organ-on-a-chip devices
allow for the culturing of an individuals cells, creating a response that models that of the
individuals body, rather than a general human response. This capability would likely be

utilized after drugs have received FDA approval, to test efficacy of the drug on a specific
individual, as shown in Figure 10.

Figure 10: The Role of Organ-on-a-Chip Devices in Individualized Treatments. In

addition to a role in the preclinical testing phase of drug development, OoC devices could
also be used in personalized clinical research. Figure reprinted from Grootaers.
The possibilities for individualized treatment has already been investigated. Y. Xu et
al. conducted a study in which the cultured cells were taken from individuals with lung
cancer during a required biopsy. This allowed for results that showed the effect of a drug in
a specific individuals body. The results of the study showed varied apoptosis rates in the
patients for each of nine drugs being investigated. In this study, results were considered in
the context of the type and stage of cancer that each patient had and allowed Y. Xu et al. to
make conclusions about the efficacy of the drugs as a function of the individuals condition
(Y. Xu).

Representation of Barriers
A struggle of in vitro testing is representation of the barriers, or partitions between fluid
compartments in the body. Barriers, while hard to model, greatly affect cell activity (Esch).
The lung alveolar-capillary barrier has been modeled successfully through the use of
OoC devices. These devices have allowed for investigation of the effects of cyclic forces due
to breathing, as shown in Figure 7 (Huh 2007, Huh 2010).
Research on drugs for central nervous system diseases, such as Alzheimer's, is
hindered by the complexities of the blood brain barrier (BBB). Several studies have created
organ-on-a-chip devices to capture the intricacies of the BBB (Helm). Microfluidic devices
have been useful for this application because the relevant size and geometries allow
endothelial cells to be exposed to physiologically relevant fluid flow. Booth et al. created a
BBB organ-on-a-chip which led to results that showed more physiologically appropriate

trans-endothelial electrical resistance as compared to in vitro tests cultured in wells (Booth).

This device, along with other OoC devices, can be used to test drugs and advance
discoveries involving the BBB.
Simulating diffusion over an appropriate time scale can be difficult with a Transwell
assay in in vitro models. OoC devices can be designed to create a model that investigates
these behaviors. Ferreira et al. conducted a study utilizing microfluidic devices to
investigate chemokinesis and chemotaxis of myoblasts across a membrane when exposed
to basic fibroblast growth factor (bFGF). Previous studies done by Ferreira et al., as well as
by others, utilized Transwell assays in similar investigations. This study determined using
finite element modelling that use of Transwell assays led to a 45% diminishment of the
bFGF gradient across the membrane within one minute. This inaccurate behavior limited
accurate investigation of chemokinesis and chemotaxis to that which occurs within the first
minute. Microfluidic devices, with the infusion of sink and source medias, allow for the
creation of a stable, linear concentration gradient. This allowed for a study of myoblast
behavior over a longer time period and yielded novel results. The use of a microfluidic
device also allowed for visualization and tracking of single cells. This enabled quantification
of individual cell velocities and aided in distinguishing between chemokinesis and
chemotaxis. Ferreira et al. was able to conclude that bFGF is only a mild chemoattractant
and that the main mechanism of myoblast migration is chemokinesis (Ferreira).

Challenges
Scaling
Organ-on-a-chip devices are useful largely due to their micro scale (Esch). However, scaling
human organs to a micro scale can be challenging.
Historically, allometric scaling has been commonly used in the design of organ-on-achip devices due to availability of data and simplicity of implementation (Abaci). In this
method, mass and cell numbers are scaled linearly while the M^() power law is used to
scale time (Guzzardi). Unfortunately, application of allometric principles can lead to
impossible proportions or misrepresentation of function in some systems (Wikswo & Curtis
2013).
Due to the limitations of allometric scaling, most OoC devices are now designed
through functional scaling. This method critically analyzes which functions of the organ
need to be represented. Scaling is adjusted, over multiple iterations if necessary, to
represent these functions appropriately. In the context of a single experiment, this method

is the most logical. However, it limits the robustness of each device and requires a new
design if the function being investigated is changed or expanded (Wikswo & Curtis 2013).
A standard set of principles, such as allometric scaling, theoretically allows for more
robust OoC models which could be used in various studies. However, allometrically scaled
models are less effective in reproducing the functions of the organ being modelled.

Measurements
Perhaps the biggest roadblock in this field is yielding useful measurements (Esch,
Maschmeyer). Most studies done with microfluidic devices only have the capability to
determine if a cell is alive (Esch, Wikswo & Block 2013). For many applications, this is
sufficient. Frequently, this is done by using confocal or fluorescence microscopy to view
stained or fluorescently labeled cells.
OoC devices do have some strengths in the area of measurements; theyre
extremely well suited to analysis through visual means, which allows for continuous real
time monitoring of individual cells. Unfortunately, there are a limited number of
conclusions that can be drawn from this type of analysis. Although cell viability can be
determined and cells can be tracked, it is difficult to do many tests that are able to perform
deeper investigation into the specific mechanisms at play.
To overcome these limitations, scientists have utilized the ability to image OoC
devices in creative ways. For example, H. Xu et al. used estrogen-sensitive green
fluorescence protein to study the effects of a chemical on estrogen production. Although
measurements of this sort are effective, they are not standardized. This leads to difficulties
in comparing studies and yields a skepticism around the reliability of results from OoC
experiments (Helm).
Trans-endothelial electrical resistance (TEER) works well as a tool for analysis and
data extraction in microfluidic devices. TEER is used to quantifiably measure the integrity of
a cell barrier and is therefore useful in study of barriers such as the BBB. It is advantageous
because it doesnt require any cell labeling and is real-time and noninvasive. Because TEER
is a standard method for in vitro models, the outputs are fairly standardized. However,
modified TEER setups for OoC devices still vary from study to study and should be
standardized to improve ease of use and promote robustness of the systems (Srinivasan).
Typical in vitro tests are limited mostly by the small volume used in microfluidic
devices. With traditional in vitro testing, small samples can be taken over time for
measurement of an analyte of interest. In an organ-on-a-chip, volumes necessary to
perform any traditional testing would cause a large enough volume decrease to affect the

experiment (Wikswo & Block 2013). Therefore, common testing such as immunostaining
can be done only at the end of an experiment (Maschmeyer).
Recent work has been dedicated to developing adapted versions of standard tests
that could be performed on OoC devices. Ghodbane et al. have developed and validated a
microfluidic device capable of multiplexing 32 parallel samples into an immunoassay
(Figure 11). This device, which serves exclusively as a tool for testing, is defined as a Labon-a-Chip, rather than an organ-on-a-chip device. However, the device provides proof of
concept for development of methods allowing for immunoassays with only several L of
volume and such methods could be incorporated into OoC devices (Ghodbane).

Figure 11: Lab-on-a-Chip Microfluidic Immunoassay Multiplexing 32 Parallel Samples.

A) Schematic including a common inlet and outlet and 32 separate ports for analysis. B)
Entire device was sized to fit on a standard microscope slide. (Ghodbane)

Design Requirements
Organ-on-a-Chip devices have many important requirements based on their intended
function. The criteria listed below are considered those necessary to allow OoC devices to
become a reliable and advantageous part of the drug discovery and development process.
First and foremost, all OoC devices need to be representative of the organ functions
that they are intended to model. The text above has confirmed this ability by outlining the
important features of OoC devices that allow them to accurately model systems and
showing evidence of successful results from experiments utilizing the devices.
In order to offer any conclusions, there must be a way to analyze results and extract
data from experiments performed in these devices. There are many visual techniques that
are well suited for OoC devices. More tests are being developed to expand the amount and
variety of analysis that can be done on experiments in OoC devices.
For OoC devices to be a feasible addition or replacement to traditional preclinical
testing, they must be economically advantageous. This criterion is met in large part

because of the ease of design and manufacturing of the devices. Furthermore, the small
scale of the devices requires smaller amounts of everything from cells and media to
reagents for testing, which further reduces costs. As seen in Figure 11, OoC devices have
the potential to be used for miniature, high throughput testing. It is widely believed that
the use of OoC as a part of the preclinical testing process could not only decrease the cost
of the preclinical testing, but also make the entire drug development process more efficient
and thus less costly (Wikswo & Block 2013, Esch).

Future Work
Much work is still necessary in order to further promote organ-on-a-chip devices as a
method for drug development testing.
Work needs to be done around measurement and analysis methods for OoC
devices. Specialized methods should be developed to account for the inability to do many
standard tests such as immunostaining. Furthermore, any new methods, as well as those
already in practice, should be standardized to allow for comparison of results from
different studies. These tasks would both advance the abilities of OoC devices and promote
them as viable tools for preclinical testing.
Further validation of these systems would make them more widely accepted as a
trusted technique. One method of validation would be to compare animal organ-on-a-chip
devices to actual animal models to confirm efficacy (Esch). OoC devices could be validated
by testing their ability to represent the effect of drugs that are already approved and on the
market. They could also be validated by being used for testing alongside clinical testing so
that in vivo results could be compared to OoC results. There are many possible avenues for
validation and further efforts in executing these possibilities should be prioritized.

Conclusion
The promise of organ-on-a-chip devices has been made clear through a multitude of
studies showing its use in drug testing. Further improvements and validation should be
prioritized so that OoC devices can be accepted as an addition to the preclinical testing
phase of drug delivery. This would require less in vitro and animal models, improve
mathematical models, speed up the drug discovery process, and decrease the cost of drug
development.

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