A microphysiological system of the human heart for drug efficacy and toxicity testing

Elliot Fisher
Under the guidance of Dr. Deok-Ho Kim, Principle Investigator
Friday June 5th, 2015
Research Proposal
Abstract. Cardiac toxicity is known to account for as much as one third of safety-related drug recalls, which
indicates an inability for current screening platforms to adequately predict compound action on human
myocardial tissue. Consequentially, the production of a microfluidic cardiac tissue mimic that is sufficiently
physiologic to produce predictive results in pharmacological studies remains a challenge in the field of tissue
engineering. The goal of this project was to develop a readily reproducible and near-physiologic microfluidic
model of the human myocardium for use in highly predictive pre-clinical drug screening. This project proposes
to develop such a cardiac model by adapting an existing commercial microfluidic chip to the growth of cardiac
tissue constructs. The chip that will be used has a cylindrical 125-micron diameter lumen for cell seeding, and
flow ports that connect to an external perfusion platform. Ultimately, a cardiac reporter cell line for the activity
of ion channels involved in action potential propagation (ArcLight-expressing human induced pluripotent stem
cell-dericed cardiomyocytes) will be used to enable the real-time assessment of engineered cardiac tissue
function. Optical measurements of the rate of action potential propagation, anisotropy, and beat frequency and
regularity will be made in conjunction with post hoc analysis of physiological maturation as indicated by
sarcomere striation, actin cytoskeleton alignment, polarized connexin43 expression, and the formation of
constructs of high cell density. The results to be collected stand to provide important information and standards
concerning the reproducible production of complete cords of myocardial cells with advanced maturation
characteristics in a microfluidic system.
Contents:
Background and significance
Problem statement and proposed solution
Previous approaches
Innovation
Preliminary Studies
Consequences of success
Real-world constraints
Scope of work
Design
Project phase overview
Research strategy
Phase 1
Phase 2
Phase 3
Key Personnel
Equipment, facilities, and resources.
Figure 3: Timeline and project phase flow chart
Table 1: Specifications, acceptance criteria, and validation tools
Appendix 1: Request for Proposal (RFP)
Appendix 2: Concept Sheet

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Background and Significance.

Problem statement and proposed solution. Cardiovascular disease is the leading cause of global mortality,
killing approximately 600,000 people each year in the United States alone - roughly 25% of all annual US
deaths [1]. Moreover, cardiovascular disease accounts for a growing number of worldwide deaths annually.
The World Health Organization (WHO) reported cardiovascular disease to have caused 16.7 million global
deaths in 2002, and anticipates cardiovascular disease to cause 22.3 million global deaths in 2030 [2].
Consequently, efforts to discover and screen new therapeutic agents capable of mitigating the effects of
cardiovascular disease, of alleviating related symptoms, or of being used in corresponding treatment regimens
is among the most pressing biomedical research currently being conducted.
Despite the need for more advanced treatments, extant drug testing and approval mechanisms are
slow, expensive, and perhaps most importantly ineffective with respect to the results they purport to
produce. Significant findings concerning adverse drug effects, particularly with respect to cardiac toxicity, have
occurred in late stage clinical studies and even during post-marketing stages of drug commercialization. This is
both tremendously expensive and poses a potential danger for patients. Given an estimated total pre-approval
cost of $802 million, the cost of late-stage drug failure during development and screening can be on the order
of tens of millions of dollars [3]. While only one out of an estimated 10,000 new chemical entities (NCEs)
investigated reaches market deployment [4], as many as 8.7% of prescription drugs are withdrawn from
worldwide markets after approval due to cardiac toxicity, costing billions of dollars annually [5], [6]. In the US
this problem is perhaps even more staggering; of the 95 compounds withdrawn from US drug markets in the
40 years preceding 2009, 19 percent were withdrawn for cardiovascular safety reasons, including 12 percent
withdrawn specifically due to proarrhythmia [6].
These statistics illustrate a patent failure of current drug screening platforms to predictively assess or
determine the arryhthmogenic effects of new drugs, which critically hampers drug discovery and production.
The FDA now requires that all new drugs be tested for arryhthmogenic potential before use in humans.
Consequently, there is a pressing urgency for biomedical research to develop more predictive ways of
screening new drugs for cardiotoxic effects.
Virtually no 3D cardiac model exists that both closely recapitulates the native conditions of the human
myocardium, and is amenable to scaling and high throughput systems. Development of a reproducible
microfluidic model of the human myocardium with advanced tissue construct maturation characteristics would
therefore be both incredibly valuable and novel. If developed, a 3D microfluidic myocardial model would have
demonstrable advantages as a drug screening platform over all existing platforms, including 2D screening
platforms, animal models, and single-cell assays.
Previous approaches. Contemporary drug screening practices and methodologies rely heavily on
animal testing. Nonhuman animal tests are known to be extremely poor proxies for determining drug effects in
humans. The FDA reported in 2004 that 92 percent of drugs that pass animal trials are later rejected when
screened in humans. Several meta-analyses have been conducted in consideration of the degree of
concordance between animal trials and human trials in drug screening [7]. Two of these studies reported ratios
of 4 out of 24 and 6 out of 114 clinical toxicities that were detectable in the animal model [8], [9]. Broadly, in
vivo screening of drugs in the animal model is expensive, slow, fails to imitate human physiology, and is often
incapable of recapitulating human disease states.
Another often-used method for drug screening is the non-cardiac multi-well platform, which has two
salient shortcomings. First, it is rendered ineffective by its inability to detect proarrhythmogenic effect, which as
discussed above accounts for a significant proportion of drug withdrawals. Second, the multi-well format, while
optimized for high throughput screening systems, does not provide the capacity to regulate or mimic many
organ-level culture conditions. By contrast, microfluidic systems enable the manipulation of culture architecture
and local environment in unprecedented ways. Researchers have long envisioned microfluidic models with the
same scalability as multi-well platforms, but with the increased manipulability and physiological relevance
offered by microfluidic systems.
Historical single-cell modeling and drug screening fail to recapitulate both the structure and function of
mature cardiomyocytes, and consequently are poor predictors of drug toxicity. Mature cardiomyocytes are
known to form highly organized confluent layers of anisotropically aligned cells in order to facilitate mechanical
contractile function. Axial alignment of cells and their myofibrils facilitates directional exertion of force and
propagation of action potentials. The electrophysiology and mechanobiology of cardiac tissue constructs are
profoundly influenced by such striated organization. Two-dimensional in vitro cardiac tissue culture models
have been developed using micro- and nano- topographical patterning techniques to fabricate elastomeric thinfilm substrates that mimic the mechanical cues imparted by the native cellular microenvironment. These

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topographically patterned substrates have been demonstrated to facilitate maturation and organization of
engineered cardiac tissue [10], but the two-dimensionality of systems relying on cellular monolayers
fundamentally limits their ability accurately recapitulate 3D tissue dynamics, mechanical activity, and
maturation states. Furthermore, it has been demonstrated that studies performed on anisotropic monolayers of
cardiomyocytes are not applicable to all forms of toxicology analysis [11].
Innovation. Our research group has spent several years developing functionally mature tissue
engineered constructs of human stem cell-derived cardiomyocytes in various systems, including
nanotopographically aligned 2D substrates and pseudo-3D multilayer stacked muscle sheets [12], 3D
nanopillar arrays [13], and 3D tissue analogues using decellularized extracellular matrix (dECM) bioink [14]. In
the course of our previous studies, we have demonstrated an ability to develop cardiac tissue constructs with
near-physiological maturation characteristics as measured by the rate of action potential propagation,
anisotropy and frequency of synchronous contractions, sarcromere striation, actin cytoskeleton alignment,
polarized connexin43 expression, and the formation of constructs of high cell density.
With the intention of applying our knowledge and techniques concerning the advanced maturation of
engineered cardiac tissue, we now proposes the development of an in vitro microfluidic model of the human
myocardium that overcomes many of the limitations of current cardiac tissue mimics. Existing platforms for
modeling human cardiac tissue are often limited either to two-dimensionality, such as the micro- and nanotopographical substrates currently being employed to produce planar constructs of anisotropic cardiac tissue
[15]; or to quasi-three dimensionality, such as microfluidic systems with minimal 3D features [16]. While these
planar platforms have been shown to induce promising tissue maturation as compared to conventional tissue
culture methods, they are fundamentally limited in their ability to recapitulate the 3D function of in vivo cardiac
tissue. While some 3D cardiac tissue models have been produced through suspension of cells in hydrogels,
their lack of sufficient mechanical cueing has led to the production of tissue constructs without functional
alignment. Other platforms have been based on the analysis of single cardiac cells [17]. Single-cell platforms
are incapable of modeling fully mature cardiac tissue because they do not allow for the extensive intercellular
biomechanical and chemical signaling interactions known to influence cardiac tissue maturity. A final limitation
of extant cardiac tissue mimics is a broad lack of reproducibility and scalability. There is still great need for the
design of a model that can be mass-produced and applied to high throughput screening applications.
Our model will employ tissue-engineered 3D architecture to recapitulate the physiological anisotropy of
native cardiac tissue, enabling cardiac cell elongation, formation of multi-cell constructs of confluent tissue, and
synchronous, directional contractile activity. The use of multi-cell tissue constructs in our model will allow for
the formation of intercellular connections and mechanical organization and elongation, characteristics that are
essential to the maturation of cardiac tissue, and which are absent in extant single-cell heart-on-a-chip models.
Preliminary Studies. Our research group has published extensively on the development of human
stem cell-derived cardiomyocyte tissue constructs with highly physiologic properties that very nearly
recapitulate cardiac tissue function in vivo [10], [13], [18], [19]. The anisotropic alignment and elongation of
native cardiomyocytes is a critical feature of cardiac tissue that is known to contribute substantially to its
contractile and electro-active functionalities [20]. The native ECM nanotopography is known to provide both
important integrin binding signal pathway activation cues and mechanical context to cardiac cells, promoting
such alignment and elongation [21]-[24]. Preliminary data gathered by our research group suggests that
human cardiomyocytes cultured in decellularized porcine cardiac ECM develop functionally mature cell sheets
as indicated by structural anisotropy, alignment and elongation, and contractile activity These results suggest
that co-injection of cardiomyocytes and dECM into the device lumen may promote more advanced maturation,
and consequently may lead to a more physiologic myocardial model.
Our groups access to a cardiac reporter cell line for the
activity of ion channels involved in action potential propagation
(ArcLight-expressing human induced pluripotent stem cell-derived
(hIPSC) cardiomyocytes) will enable real-time optical observation of
electrical conductivity in living tissue constructs, a practical proxy for
the analysis of tissue function and contractile activity. Using
ArcLight-mediated optical observations, we will be able to interrogate
Figure 1: The prototype microfluidic chip to be
optimized for cardiac tissue constructs (Nortis)
conduction velocity, beat frequency, unified contractile activity, and
signal propagation.
The prototype microfluidic chips we will use are cast in Polydimethylsiloxane (PDMS) from precision-milled
wax molds. They have four ports: two inlets and two outlets. Either inlet or outlet pair can be used for perfusion of
blood surrogate media. One pair provides media flow in the luminal direction, while the other provides flow in the
transverse direction through the collagen. The device has a 42-microliter-volume chamber that is filled with type-I

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collagen in preparation for cell seeding. The collagen is injected through a side-port and is set around a smalldiameter glass capillary. Once the collagen is gelled, the glass capillary is removed, leaving a micro-scale lumen in
the collagen for cell seeding (Figure 1).
Preliminary experiments have been conducted using GCaMP3-expressing Rockefeller University
Embryonic Stem cell (REUS2)-derived cardiomyocytes. In the first of these preliminary experiments, the sell seeding
lumen was molded in type-I collagen with a density of 3 mg/mL to have a diameter of 125 microns. The lumen
received a surface treatment of fibronectin at 50 g/mL to promote cell adhesion and medium was perfused through
the collagen matrix perpendicular to the lumen at a flow rate of 0.5 microliters per minute. This low flow rate was
presumed to be insufficient to provide significant sheer or normal stress mechanical cues. Cardiomyocytes were
seeded under these conditions at a density of 20 million cells per mL. Cells cultured under these conditions showed
promising survivability and maturation.
Bright-field microscopy indicated partial
cell aggregation and some signs of
mature morphology, such as cell
elongation around the periphery of the
tissue constructs (Figure 2a). Observable
contractile synchronicity emerged within
the first five days of culture. Green
Fluorescent Protein (GFP) imaging of the
GCaMP3-expressing contractile tissue Figure 2: (a) Left: Bright field, and (b) Right: GFP imagery of a
constructs showed that most cells in the cardiomyocyte tissue construct during contraction within the
tissue constructs fluoresced at the same 125-micron lumen of the prototype device.
time, indicated that calcium signaling by release of stores from the sarcoplasmic reticula was occurring in unison,
another promising characterization (Figure 2b). Beat frequency of these preliminary models was measured at
approximately 0.2 Hz.
A higher beat frequency, improved cell-lumen attachment, increased cell-density, and more robustly mature
morphologies will need to be attained. All of these goals are within the design scope of this proposal. These
preliminary results strongly indicate the feasibility of integrating cardiac cell lineages with this existing microfluidic
platform.
Consequences of success. We expect that our model will be able to detect drugs with known cardio-toxic
effect. Furthermore, due to the increased physiological characterization and advanced maturation states of the
tissue constructs it contains, we anticipate that out model will outperform both the animal model and existing in vitro
systems in terms of concordance with published data from human trials, and in terms of lower dose response.
Real-world constraints.
Ethical and social. The cardiomyocyte cells that this proposal seeks to employ are derived from a line of
Rockefeller University Embryonic Stem Cells (REUS2). Embryonic stem cells of this origin are sourced from a deidentified frozen embryo that was donated with informed consent for research purposes. There is no catalogued
information that can be used to identify the original embryonic tissue donor.
Economic. The research and development of this novel microfluidic drug-screening platform will be
contingent on the availability of the stably-transfected MP3-expressing RUES2 human embryonic pluripotent stem
cell derived cardiomyocytes as well as other stem cell-derived cell lineages. The differentiation and transfection
protocol for these stem cell lineages takes on the order of 30 days, requiring both extensive resources and labor
hours. Consequently, great care will have to be taken in the execution of this project such that cells are only used
when experimentally necessary. For this reason, some optimization steps may be performed using other more
readily available cell lineages such as C2C12 myoblasts. With the ultimate goal of producing a commercializable
product, care will have to be taken to keep assay costs within or below the relevant market ranges. Because this
project will be completed using an existing commercial prototype, commercial translation will be relatively
inexpensive. Much of the necessary infrastructure for this process already exists in the facilities of our close
commercial collaborator, Nortis Bio.
Legal and regulatory. The International Life Sciences Institute (ILSI) has recently developed and publicized
an initiative called the Comprehensive In Vitro Proarrhythmia Assay (CIPA). Currently, FDA safety guidelines E14
and ICH S7 a/b guidelines govern preclinical safety screening of pharmaceuticals. The CIPA initiative seeks to
supplant or modify those guidelines. The research and development efforts conducted within the scope of this
project will seek to produce an in vitro microfluidic cardiac drug screening platform in accordance with ILSI CIPA.

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Scope of Work.
Design. The ultimate goal of this project is to develop a readily reproducible and near-physiologic microfluidic model
of the human myocardium for use in highly predictive pre-clinical drug screening. Organ-on-a-chip microfluidic
models of various tissue types and cell lineages are currently being developed for use in pharmacological studies
with improved throughput, cost-effectiveness, and predictive capacity. However, no cardiac models have been
produced under this paradigm that recapitulate native cardiac tissue accurately enough to be used in drug screening
applications.
Cardiac toxicity is known to account for as much as one third of safety-related drug recalls [25], which
indicates an inability for current screening platforms to adequately predict compound action on human myocardial
tissue. Consequentially, the production of a microfluidic cardiac tissue mimic that is sufficiently physiologic to
produce predictive results in pharmacological studies remains a challenge in the field of tissue engineering.
However, our research group has previously demonstrated the ability to produce cardiac tissue constructs of
advanced maturation states in various 2D and 3D systems. We have also demonstrated an ability to robustly
characterize the degree of physiological resemblance and functionality of engineered cardiac tissues by observing
their structural and anisotropic properties [10], [12], [18], [19], [26]-[28]. This expertise can be readily applied to the
development of a microfluidic cardiac model.
Engineering design standards. The policy environment surrounding preclinical screening of drugs for
cardiotoxicity is developing rapidly. The International Life Sciences Institute (ILSI) has been developing an initiative
called the Comprehensive In Vitro Proarrhythmia Assay (CIPA). The initiative seeks to eliminate FDA safety
guidelines E14 and modify ICH S7 a/b guidelines, both of which currently govern preclinical safety screening of
pharmaceuticals. The goal of this project is to produce an in vitro microfluidic cardiac drug screening platform with
engineering design standards in accordance with ILSI CIPA such that its relevance and policy compatibility in the
emerging paradigm of advanced preclinical drug screening is assured.
This project will be executed using a commercially available prototype microfluidic organ-on-a-chip platform,
which has been used by research groups for the generation of model tissues comprised of many non-cardiac cell
types, including kidney, liver, brain, testis, and microvascular cells. The integration of our research groups
experience in cardiac tissue engineering and of this existing platform stands to fill a critical void in our current ability
to perform predictive preclinical drug screening using cardiac tissue mimics. To address this gap in technology and
knowledge, our group will use cardiac tissue engineering techniques developed in our lab in three phases:
Phase 1. Develop and optimize a microfluidic model of the human myocardium by modification of an
existing commercial microfluidic platform using cardiac tissue engineering techniques. The existing
commercial microfluidic system has been previously implemented with non-cardiac cell lineages. We anticipate that
modification will be necessary in order to adapt the system to the growth of cardiac tissue constructs. Studies will be
conducted to optimize a) the platforms mechanical parameters, including medium perfusion flow rate and continuity;
b) the system microenvironment using lumen surface treatment and cell co-injection with decellularized cardiac
extracellular matrix (dECM); and c) protocols associated with cardiac cell culture and seeding. Use of a cardiac
reporter cell line for intracellular voltage changes (ArcLight hIPSC cardiomyocytes), will enable the real-time
assessment of engineered cardiac tissue function during optimization studies. Optical measurements of the rate of
action potential propagation, anisotropy, and beat frequency and regularity will be made in conjunction with post hoc
analysis of physiological maturation as indicated by sarcromere striation, actin cytoskeleton alignment, polarized
connexin43 expression, and the formation of constructs of high cell density. The results collected will provide
important information and standards concerning the reproducible production of complete cords of myocardial cells
with advanced maturation characteristics in a microfluidic system.
Phase 2. Validate our microfluidic model of the human myocardium by characterizing its predictive capacity
in drug cardiotoxicity screening. It is our intention to demonstrate that our tissue engineered microfluidic system
could be feasibly employed in predictive preclinical screening of drugs for arrhythmogenic potential. A baseline for
tissue performance will be defined in terms of electrophysiological activity as determined by optical measurement
and computational analysis. Mature cardiac tissue constructs will be perfused with drugs possessing either
arrhythmogenic or non-arrhythmogenic properties, and compared to the baseline to verify the systems ability to
detect drug-induced alterations to functional phenotype. Results will then be compared to current preclinical screens
to determine whether the developed microfluidic system has improved sensitivity as compared to conventional drug
efficacy and toxicity assays.
Phase 3. Prepare model for commercialization by reducing costs and standardizing production protocol.
After optimization and validation steps have been completed, material and assembly costs and well as scalability
and manufacturing amenability will be assessed. Efforts will be made to minimize the cost added to the existing
commercial platform while preserving the functional standards developed in phases 1 and 2.

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Research Strategy.
Phase 1. Develop and optimize a microfluidic model of the human myocardium by modification of an
existing commercial microfluidic platform using cardiac tissue engineering techniques. Rationale: Phase 1
is intended to yield and characterize a stable and reproducible model. Optimization steps conducted in pursuit of this
phase will ultimately enable the production of a myocardial model that integrates critical maturation and functional
characteristics of native cardiac tissue in vivo in a way that no prior art has been able to achieve. We anticipate that
modification of the existing microfluidic system will be necessary to achieve such results, particularly with respect to
mechanical parameters such as system microenvironment which provides important mechanical cues to enclosed
tissue constructs.
Experimental Approach. A prototype microfluidic chip and perfusion platform system designed for use with noncardiac cell lineages is commercially available (Nortis). The current chip housing is produced using PDMS molding
from a precision-milled wax master. This process is readily manipulable. If large-scale structural amendments are
necessary to the chip for the optimal culture of cardiac cells, such changes can be implemented readily. The current
chip features four microfluidic channels: two inlet ports and two outlet ports. One pair of ports supplies the chip
lumen, and the other supplies the surrounding collagen chamber. Two septum plugs on the top of the chip provide
syringe access to the lumen. All microfluidic channels are equipped with bubble-traps to prevent the interruption of
flow due to the introduction of small quantities of air. The current dimensional specifications of the chips are as
follows: the collagen chamber has a volume of 42 L; the lumen has a diameter of 125 m, and a volume of 70 nL.
The lumen is produced by molding type-1 collagen at a density of 3 g/mL around a glass capillary of the
appropriate diameter, which is then removed prior to cell seeding.
ArcLight-expressing hIPSC cardiomyocytes will be differentiated and cryopreserved for later use.
Preliminary studies suggest that approximately 75,000 cells are required per chip per seeding instance. In order to
facilitate early-stage experiments on the order of ten chips being tested in parallel, cells will be cryopreserved in
aliquots of one million cells. In advance of performing seeding into the microfluidic lumen, cells will be carried
through a thawing protocol and plated in a standard petri-dish format for at least one day and for no more than three
days. The blood surrogate medium used to maintain cell cultures will be Roswell Park Memorial Institute (RPMI)
medium (Life Technologies) supplemented with L-glutaimine, B27 supplement and 1% penicillin-streptomycin
solution. Before cell seeding, chips will be primed with gas-and-temperature-equilibrated medium for one hour. Cells
that survive the freeze-thaw cycle will be resuspended and slowly injected into the microfluidic lumen using a sideaction syringe through the seeding septum of the microfluidic chip. No medium perfusion will be applied to the chip
for the first hour of cell culture in order to promote cell attachment to the lumen. One hour after seeding, growth
medium will be perfused into the chips at a rate of 0.5 L/min in the transverse direction, through the collagen
matrix. Perfusion of media will be achieved by attachment of the microfluidic chips to a prototype perfusion platform
produced by the same commercial supplier (Nortis). This platform is designed to lock the microfluidic system in
place to prevent inadvertent discontinuity of flow pressure due to jostling or lengthy tubing. The perfusion platformmounted chips will be stored in cell-culture incubators at 37 oC and 5% CO2, and will be driven by external incubator
gas pumps with programmable pressure parameters.
Imaging. Fluorescent imaging of calcium transients will be acquired with a 488 blue light laser integrated
with a Zeiss LSM 510 confocal microscope system using Plan Apo 10x/0.5 NA objective. Conventional bright field
microscopy will be used to monitor cell and tissue construct morphologies and maturation. Constructs will be
evaluated daily to monitor development and in order to determine the length of culture necessary for the formation of
optimally mature and functional tissues.
Cardiomyocyte cell lineages are known to be highly sensitive to mechanical cues imparted by their
microenvironment, and by extension, those imparted by their culture substrata [12], [18], [26], [29]. The primary
mechanical cues of concern in this system are (1) cell density, (2) the diameter of the cell-seeding lumen, and the
(3) characteristics of the lumen microenvironment. These will be our first three targets for experimental manipulation
and optimization for cardiac cell culture. Experimental conditions will be analyzed in groups of four chips cultured in
parallel perfusion platforms.
(1) Cell seeding densities and injection methods will be varied with a target post-seeding density in the
range of literature-reported 2.4 0.2 10^8 cells/cm3 for physiologic cardiomyocytes [30]. The post-seeding
densities given by single- and multi-puncture injections will be compared. Cell densities will be determined using
nuclear staining and quantification via confocal imaging. Live/dead assays will be performed to quantify cell survival
in the chips.
(2) Glass capillaries for collagen matrix molding are available in a 125-micron diameter and a 145-micron
diameter sheathing a 65-micron inner capillary. Thus, we have the capacity to produce chips with 125-micron
diameter lumens, and those with 145-micron diameter lumens with 65-micron diameter glass capillaries at the
center. Maturation characteristics and general structural morphologies of tissue constructs grown in chips with large
and small diameter lumens will be compared. Preliminary experimentation using the sheathed dual-capillary

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approach has been attempted using the same broad seeding and culture protocols as articulated above. Cells
cultured in these systems have been observed to develop tissue structures of higher density and confluence than
those seen in chips with 125-micron lumens. By day 7, contractile synchronicity was observed spanning distances
on the order of 2000 microns in the luminal direction, a significant improvement in signal conduction distances over
those observed in 125-micron diameter chips. These contractions were observable at a frequency of approximately
0.1 Hz using both bright field microscopy (Figure 3a) and GFP imaging of GCaMP3-indicated release of calcium
stores (Figure 3b).
(3) The surface characteristics of the lumen are also anticipated to influence the maturation characteristics
of tissue constructs. Both PLLand Fibronectin-treatment of cell
culture substrata are conventional
and well-documented approaches
to promoting cell adhesion [31],
[32]. Chips with bare type-1
collagen luminal surfaces (as
molded) will be compared to those
with fibronectin-treated luminal
surfaces and to those with poly-L- Figure 3: (a) Left: Bright field, and (b) Right: GFP imagery of a
lysine
(PLL)-treated
luminal cardiomyocyte tissue construct surrounding a 65-micron glass capillary
surfaces.
To
promote
the during contraction in the modified prototype device.
formation of a solid, lumen-filling
chord of myocardial tissue, we will also investigate suspending cells in porcine-derived cardiac ECM (2% solution)
before injection. Before co-injection with cells, phase contrast microscopy will be used to verify that no undesirable
fluid paths or distortions arise from the use of dECM hydrogel in the lumen.
Expected Results, Interpretation, Possible Pitfalls.
Key objective: Reproducibly generate complete cords of myocardial cells spanning 90 percent of the lumen
in the microfluidic chips with one-week viability. We expect that the chips with a 65-micron-diameter lumen will
outperform those with a 125-micron-diameter lumen because a narrower lumen will provide a more readily
perceptible anisotropic mechanical cue on the cellular level. We also expect surface treatment to positively influence
cell adhesion and subsequent maturation of tissue constructs. For the purposes of this investigation, we will define
physiological cardiac maturation as ultrastructural organization of the sarcomere and alignment of myofibrils
(measured using immunocytochemistry, shorter action potential duration (measured by optical proxy through realtime confocal imaging of ArcLight fluorescence), hypertrophy (measured with phase-contrast microscopy),
cardiomyocyte alignment (measured by immunocytochemical staining for actin filaments), and formation of
intercalated disks (measured by immunocytochemical staining for connexin-43 and N-cadherin).
It is possible that direct injection of cells into the lumen will lead to poor nutrient diffusion to the core of
cylindrical tissue constructs. This will be of more concern in large-diameter-lumen chips, but could still present
issues of internal necrosis in the small-diameter-lumen chips. Furthermore, it is possible that even the smalldiameter lumen will not provide a sufficiently strong mechanical cue to promote cardiomyocyte structural elongation
and anisotropy. These problems could be addressed by first investigating seeding cells only around the perimeter of
the lumen using sheathed molding capillaries, and second by switching from transverse flow to luminal flow.
Seeding only the perimeter of the lumen would improve nutrient diffusion and media flow, and luminal flow would
provide an additional anisotropic mechanical cue to promote cardiac tissue organization and maturation.
Additionally, achieving cell density high enough to closely mimic physiological conditions may prove challenging. If
this is the case, an alternate seeding protocol may be employed, wherein the downstream channel of the lumen
would be blocked during slower cell injection, and luminal flow would be minimized. These adjustments would be
made to limit the wash-through of cells before they have had sufficient time to form initial attachments to the lumen
surface and to each other. After attachments have been established, the capillary would be fully removed and flow
could be directed in the luminal direction to resume mechanical cueing.
Phase 2. Validate our microfluidic model of the human myocardium by characterizing its predictive capacity
in drug cardio-toxicity screening. Rationale: This phase is designed to validate our systems ability to model
cardiac function in response to perfusion with compounds of known cardio-toxic effect. Based on results previously
reported by other groups demonstrating measurement of cardiomyocyte functionality with biomedical
microelectromechanical (BioMEMS)-based microfluidic platforms [33]-[35], we are confident that our model will be
employable as a predictive drug-screening platform. Furthermore, our models feasibility is strengthened by the
integration of ArcLight-expression, which has been shown to be a highly reliable means of producing data
concerning the contractile activity and excitability of engineered cardiac tissues [36]. The use of optical and

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computational analysis, as enabled by the inclusion of ArcLight, will leave our system well suited to integration into
multi-scale drug screening applications and assay systems, ranging from single analysis to mass production and
rapid parallel analyses.
Experimental Approach. We will first establish baseline characteristics of maturation and cardiac tissue function in
our system. Tissue performance will be measured and quantified using both bright field microscopy and GFP
imaging using a 488 nm blue laser in a live-cell temperature, humidity, and carbon dioxide controlled confocal
microscope system. The latter will lend insight into electrophysiological activity of our tissue constructs by causing
ArcLight reporter molecules expressed by our cardiomyocyte lineage to fluoresce during electrical signaling events.
Analysis of cultured tissue constructs will be conducted according to the general protocols and timeframe regimes
outline in Phase 1. Initial validation studies will be conducted sample-by-sample using manual analytical methods.
We will work to develop automated high-throughput sample processing infrastructure based on image processing
and computational analysis. Contractile activity of cell constructs and encompassing scaffold materials will be
recorded using a phase-contrast microscope equipped with a digital video recorder capable of capturing between 10
and 20 frames per second. An image processing algorithm will be written in MATLAB to measure local displacement
of the cell construct from sequential frames in the contraction video. Each image will then be divided into equal
blocks of 16 by 16 pixels and analyzed using a search algorithm that determines the x-axis and y-axis displacement
of each block between sequential frames. Each frame of a contraction cycle will then be represented by a
displacement vector map and a displacement amplitude contour map. In order to quantify synchronized contraction,
autocorrelation can be computed for the calculated displacement vectors in a number of regions within the construct
and plotted as a function of time. Comparison of the correlation co-efficient between separate curves will then give
an indication of synchronicity. Each specimen will be analyzed in this manner for 5 seconds of recorded video.
Using these 2 techniques, at least 20 chips will be tested during this phase in order to gain strong indications as to
the reliability of the functional performance of cells seeded in this microenvironment and our ability to effectively
quantify this performance.
We will then compare baseline characteristics to system responses to drugs with known cardio-active or
cardio-toxic effects in order to validate the systems ability to report expected changes in functional phenotype of
engineered tissues. Parallel chips will be perfused at incremented compound concentrations to determine minimum
detection concentration and to relate concentration and functional response. During drug administration,
observations will be recorded under a temperature and atmosphere regulated live-cell confocal microscope system.
Potential drugs to be analyzed include the following: terfenadine and cisapride, known arrhythmogenic (human
Ether--go-go-Related Gene) hERG blockers that are difficult to detect using in vivo animal screens such as the
Purkinje fiber assay and in vitro animal screens such as the Langendorff assay; moxifloxacin, a hERG blocker used
as a positive control in clinical thorough QT studies; terodiline, a mixed ion channel blocker known to produce QT
interval prolongation; verapamil, a mixed ion channel blocker that does not prolong the QT interval; flecainide and
quinidine, which are sodium channel blockers; nitrendipine, a calcium channel blocker; ranolazine, a complex ion
channel block that prolongs the QT interval but is approved for use in humans; and ranolazine, a PDE5 inhibitor
known to prolong the QT interval in humans without inhibiting hERG.
Expected Results, Interpretation, Possible Pitfalls.
We expect that our model will be able to detect drugs with known cardio-toxic effect. Furthermore, due to the
increased physiological characterization and advanced maturation states of the tissue constructs it contains, we
anticipate that out model will outperform both the animal model and existing in vitro systems in terms of
concordance with published data from human trials, and in terms of lower dose response.
However, it is possible that despite advanced maturation states of our cardiac tissue constructs, the systems ability
to detect and report arrhymogenic effect will not be as expected or may conflict with known characterizations of
cardio-active drugs. Co-culture of endothelial cells may be an important facet of predictive drug screening models
because it would enable the analysis of drug metabolic passage through the endothelium. We choose not to include
endothelial co-culture in our initial model in pursuit of the production of the simplest highly physiologic and predictive
model possible. However, if predictive results cannot be attained via the monoculture of mature cardiomyocytes, we
will investigate the integration of an endothelial cell layer into our model. Such integration would be achieved using
multi-step cell seeding protocols, wherein endothelial cells would be seeded in advance of the introduction of
cardiomyocytes using a sheathed molding capillary. Endothelial cells would be cultured on the surface of the lumen
forming a hollow endothelial tube, and cardiomyocytes would then be seeded at the core of the lumen. Transverse
luminal flow would then be used to ensure that as drug-carrying media passed through the tissue constructs, they
would have to pass through the model endothelium before reaching cardiomyocyte cells. Successful implementation
of similar endothelial cell systems with other cell types has been reported in previous research [37]. This precedent

Page 8

suggests that such integration with cardiac cell lineages is feasible. If necessary, the integration of cardiac
fibroblasts may also be explored to improve tissue cohesiveness.
Phase 3. Prepare model for commercialization by reducing costs and standardizing production protocol.
This phase will be entered pending the completion of Phases 1 and 2. The goal of this phase will be to minimize the
costs associated with the production of the established cardiac microfluidic model such that it can be scaled, easily
mass produced, marketed, and commercialized. This will be a product development phase with a focus on industrial
and economical considerations as opposed to technical specifications and research efforts.
Practical Approach. After the proposed microfluidic cardiac model has been functionally optimized, characterized,
and evaluated for sensitivity to the detection of arrhymogenic compounds, materials will be quantified. Both
materials costs and assembly costs will be tabulated to determine the marginal cost of producing one unit assuming
the at-cost purchase of the base product existing microfluidic chip. Costs will be assessed in United States Dollars
assuming domestic purchase of supplies and reagents. For those reagents or supplies that are not commercially
available or that require technical expertise to produce, labor costs and raw materials will be used as proxies to
compute materials or reagent costs. The costs associated with the optimized product will be compared to the costs
estimated for a minimum viable commercial product. The minimum viable commercial product will be assembled
and iterated through abbreviated proof-of-function studies as articulated in Phases 1 and 2. If functional, this version
of the model will be accepted as the final deliverable under the scope of this project. If not functional, revised
minimum viable commercial products will be designed, analyzed for cost, and tested accordingly until a functional
version is developed.
Expected Results, Interpretation, Possible Pitfalls. We anticipate that the materials expenses associated with
the development of our microfluidic cardiac model will be negligible. Simple reagents such as collagen type I and
dECM gels or ECM proteins for surface treatment are anticipated to be the only costs beyond the costs of the
existing commercial microfluidic housing and platform. If this is the case, this Phase will serve as a quantification
step in the development process, translating technical developments into economic quantities. If expenses are
determined to be more than the anticipated marginal increase over those of the existing chips, the possibility of
using alternative materials or fabrication procedures to minimize costs will be assessed. Cost-benefic analysis will
be used to determine whether minimum-cost production schemes are worth any perceived deficits in functional
performance.
Key personnel. This project will be completed by Elliot Fisher in close collaboration with Jasmine Rinnofer, a
visiting M.S. student from the University of Applied Sciences, Vienna, Austria. Guidance and mentorship will be
provided by Dr. Alec Smith, a post-doctoral scholar in our research group, Magnus Koller, a research scientist
employed by Nortis Bio, and Dr. Deok-Ho Kim, the principle investigator our research group.
Equipment, facilities, and resources. The research efforts articulated above will be conducted in Dr. Deok-Ho
Kims Research Group in the Brotman Building in South Lake Union, Seattle, Washington. Our development efforts
will be bolstered by our access to three key resources: (1) First, our access to a human stem cell-derived
cardiomyocyte cell lineage with a genetically encoded fluorescent calcium indicator will allow for the optical
characterization of tissue performance in real-time. (2) Second, our model will benefit from our access to an existing
microfluidic platform produced using commercial manufacturing workflows and infrastructure. Nortis Bio will be
contributing prototype microfluidic chips and equipment free of charge in the spirit of academic collaboration. The
reproducibility of our model is of great import, and so working with a commercial collaborator puts us at an
advantage over exclusively academic research groups, particularly in our ability to develop disposable devices that
could be mass-produced or adapted to existing high-throughput analytical systems or multi-organ devices.
Additionally, we will use an existing prototype microfluidic perfusion platform allowing for medium perfusion for
maintenance of cultures, supply and removal of experimentally applied compounds, manipulation of flow rate for
modulation of sheer stress, and control of mechanical cues to influence cell mechanobiology and maturation. (3)
Third, our labs production of dECM will allow us to investigate the benefit of suspending cells in native cardiac
matrix before injection into the device lumen. dECM has been reported to promote tissue maturation due to
improved activation of maturation signaling pathways through cardiac-specific integrin binding compatibility with
ECM ligands [21]-[23], [38], [39]. We anticipate that dECM will further benefit tissue maturation due to mechanical
integrity it affords as an element of the cell micro- and nano-environment. Ultimately, these resources and concepts
leave us well positioned to produce cardiac tissue constructs of much improved physiological relevance as
compared to prior art.

Page 9

Figure 3: Timeline and Diagrammatic depiction of the iterative design process. Each phase will reference and
be contingent upon the standards and outcomes established by previous phases.
Table 1: Specifications, acceptance criteria, and validation tools
Phase
Specification
Acceptance Criteria
Cell dense retention post seeding
(80% of cells remain in lumen); 0%
Perfusion flow rate
luminal necrosis due to inadequate
media supply
20 million/mL injection without
Cell density
device rupture
Standardized by characterization
Action potential
across 10 trails with minimal
propagation rate
variance
80% of luminal cells exhibiting
Anisotropy
anisotropic morphology
Phase 1
Beat frequency

Phase 2

Phase 3

Page 10

Sarcromere striation
Actin cytoskeleton
alignment
Polarized connexin43
expression
Improved sensitivity to
arrhythmogenic
compounds as compared
to animal model
Improved sensitivity to
arrhythmogenic
compounds as compared
to existing in vitro models
Additional cost

1Hz +/- 0.2 Hz

80% of luminal cells exhibiting

cardiac characteristic phenotypes
and morphology

Our model produces a positive

result where the other model does
not when screening a drug with
known arrhymogenic effect

Less than $40 per assay addtl

Validation Tool
Live-cell bright field
microscopy
Live-cell bright field
microscopy, hemocytometer
Zeiss LSM 510 confocal
microscope, ArcLight
Reporter fluorescence
Live-cell bright field
microscopy
Zeiss LSM 510 confocal
microscope, ArcLight
Reporter fluorescence,
Live-cell bright field
microscopy
Post hoc
immunofluorescence
imaging using conjugated
antibodies
Zeiss LSM 510 confocal
microscope, ArcLight
Reporter fluorescence,
Live-cell bright field
microscopy, post hoc
immunofluorescence
imaging using conjugated
antibodies
DM and DL accumulation
costing accounting methods

References:
[1]
S. L. Murphy, J. Q. Xu, and K. D. Kochanek, Deaths: final data for 2010, National vital statistics reports, vol.
61, no. 4, pp. 1118, 2013.
[2]
C. D. Mathers and D. Loncar, Projections of Global Mortality and Burden of Disease from 2002 to 2030,
PLoS Med, vol. 3, no. 11, p. e442, Nov. 2006.
[3]
J. A. DiMasi, R. W. Hansen, and H. G. Grabowski, The price of innovation: new estimates of drug
development costs, J Health Econ, vol. 22, no. 2, pp. 151185, Mar. 2003.
[4]
H. Willenbring, L. P. Lee, and K. E. Healy, Human induced pluripotent stem cell-based microphysiological
tissue models of myocardium and liver for drug development, stem cell research & , 2013.
[5]
M. Fung, Evaluation of the characteristics of safety withdrawal of prescription drugs from worldwide
pharmaceutical markets-1960 to 1999, Drug Information Journal, vol. 35, no. 1, pp. 293317, 2001.
[6]
J. P. Piccini, D. J. Whellan, B. R. Berridge, J. K. Finkle, S. D. Pettit, N. Stockbridge, J.-P. Valentin, H. M.
Vargas, and M. W. Krucoff, Current challenges in the evaluation of cardiac safety during drug development:
Translational medicine meets the Critical Path Initiative, American Heart Journal, vol. 158, no. 3, pp. 317
326, Sep. 2009.
[7]
H. Olson, G. Betton, D. Robinson, K. Thomas, A. Monro, G. Kolaja, P. Lilly, J. Sanders, G. Sipes, W.
Bracken, M. Dorato, K. Van Deun, P. Smith, B. Berger, and A. Heller, Concordance of the toxicity of
pharmaceuticals in humans and in animals, Regul. Toxicol. Pharmacol., vol. 32, no. 1, pp. 5667, Aug.
2000.
[8]
R. Heywood, Clinical Toxicity--Could it have been predicted? Post-marketing experience. Animal Toxicity
Studies: Their Relevance for , 1990.
[9]
G. N. Fracchia, C. Spriet-Pourra and M. Auriche Worldwide Product Safety & Epidemiology The withdrawal
of a drug from the market for safety reasons is not a rare phenomenon. , European Medicines Research:
Perspectives in , 1994.
[10]
D.-H. Kim, E. A. Lipke, P. Kim, R. Cheong, S. Thompson, M. Delannoy, K.-Y. Suh, L. Tung, and A.
Levchenko, Nanoscale cues regulate the structure and function of macroscopic cardiac tissue constructs.,
Proc. Natl. Acad. Sci. U.S.A., vol. 107, no. 2, pp. 565570, Jan. 2010.
[11]
T. Kaneko, K. Kojima, and K. Yasuda, An on-chip cardiomyocyte cell network assay for stable drug
screening regarding community effect of cell network size., Analyst, vol. 132, no. 9, pp. 892898, Sep. 2007.
[12]
A. Jiao, N. E. Trosper, H. S. Yang, J. Kim, J. H. Tsui, S. D. Frankel, C. E. Murry, and D.-H. Kim,
Thermoresponsive Nanofabricated Substratum for the Engineering of Three-Dimensional Tissues with
Layer-by-Layer Architectural Control, ACS Nano, vol. 8, no. 5, pp. 44304439, May 2014.
[13]
D.-H. Kim, P. Kim, K. Y. Suh, S. K. Choi, S. H. Lee, and B. Kim, Modulation of Adhesion and Growth of
Cardiac Myocytes by Surface Nanotopography. IEEE, 2006, pp. 40914094.
[14]
F. Pati, J. Jang, D.-H. Ha, S. W. Kim, J.-W. Rhie, J.-H. Shim, D.-H. Kim, and D.-W. Cho, Printing threedimensional tissue analogues with decellularized extracellular matrix bioink, Nat Commun, vol. 5, Jun. 2014.
[15]
H. N. Kim, A. Jiao, N. S. Hwang, M. S. Kim, Do Hyun Kang, D.-H. Kim, and K.-Y. Suh, Nanotopographyguided tissue engineering and regenerative medicine, Advanced Drug Delivery Reviews, vol. 65, no. 4, pp.
536558, Apr. 2013.
[16]
S. G. Hong, B. R. Conklin, L. P. Lee, A. Stahl, and K. E. Healy, Design, fabrication and characterization of
microphysiological systems to study drug toxicity in cardiac and adipose tissue, abstracts.biomaterials.org
.
[17]
W. Cheng, N. Klauke, H. Sedgwick, G. L. Smith, and J. M. Cooper, Metabolic monitoring of the electrically
stimulated single heart cell within a microfluidic platform, Lab on a Chip, vol. 6, no. 11, pp. 14241431, 2006.
[18]
D.-H. Kim, Kshitiz, R. R. Smith, P. Kim, E. H. Ahn, H. N. Kim, E. Marbn, K.-Y. Suh, and A. Levchenko,
Nanopatterned cardiac cell patches promote stem cell niche formation and myocardial regeneration, Integr.
Biol., vol. 4, no. 9, pp. 10191033, 2012.
[19]
H. N. Kim, A. Jiao, N. S. Hwang, M. S. Kim, D.-H. Kang, D.-H. Kim, and K.-Y. Suh, Nanotopography-guided
tissue engineering and regenerative medicine, Advanced Drug Delivery Reviews, vol. 65, no. 4, pp. 536
558, Apr. 2013.
[20]
H. N. Kim, D.-H. Kang, M. S. Kim, A. Jiao, D.-H. Kim, and K.-Y. Suh, Patterning Methods for Polymers in
Cell and Tissue Engineering, Ann Biomed Eng, vol. 40, no. 6, pp. 13391355, Jan. 2012.
[21]
S. BADYLAK, D. FREYTES, and T. GILBERT, Extracellular matrix as a biological scaffold material:
Structure and function, Acta Biomaterialia, vol. 5, no. 1, pp. 113, Jan. 2009.
[22]
T. Hoshiba, H. Lu, N. Kawazoe, and G. Chen, Decellularized matrices for tissue engineering,
www.expertopin.com/ebt, vol. 10, no. 12, pp. 17171728, Nov. 2010.
[23]
S. F. Badylak, The extracellular matrix as a scaffold for tissue reconstruction, Seminars in Cell &
Developmental Biology, vol. 13, no. 5, pp. 377383, Oct. 2002.
[24]
Cell Biology of Extracellular Matrix, 1991.
[25]
L. Guo, R. M. C. Abrams, J. E. Babiarz, J. D. Cohen, S. Kameoka, M. J. Sanders, E. Chiao, and K. L. Kolaja,
Estimating the Risk of Drug-Induced Proarrhythmia Using Human Induced Pluripotent Stem Cell-Derived

Page 11

[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]
[39]

Page 12

Cardiomyocytes, Toxicol. Sci., vol. 123, no. 1, pp. 281289, Sep. 2011.
Kshitiz, J. Park, P. Kim, W. Helen, A. J. Engler, A. Levchenko, and D.-H. Kim, Control of stem cell fate and
function by engineering physical microenvironments, Integr. Biol., vol. 4, no. 9, pp. 10081018, 2012.
H. S. Yang, N. Ieronimakis, J. H. Tsui, H. N. Kim, K.-Y. Suh, M. Reyes, and D.-H. Kim, Nanopatterned
muscle cell patches for enhanced myogenesis and dystrophin expression in a mouse model of muscular
dystrophy., Biomaterials, vol. 35, no. 5, pp. 14781486, Feb. 2014.
D. H. Kim, P. K. Wong, and J. Park, Microengineered platforms for cell mechanobiology, Annual review of
, 2009.
D. H. Kim, P. P. Provenzano, and C. L. Smith, Matrix nanotopography as a regulator of cell function, The
Journal of cell , 2012.
M. Mollova, K. Bersell, S. Walsh, J. Savla, L. T. Das, S.-Y. Park, L. E. Silberstein, C. G. dos Remedios, D.
Graham, S. Colan, and B. Khn, Cardiomyocyte proliferation contributes to heart growth in young humans.,
Proc. Natl. Acad. Sci. U.S.A., vol. 110, no. 4, pp. 14461451, Jan. 2013.
G. X. Wang, X. Y. Deng, C. J. Tang, L. S. Liu, L. Xiao, L. H. Xiang, X. J. Quan, A. P. Legrand, and R.
Guidoin, The adhesive properties of endothelial cells on endovascular stent coated by substrates of poly-Llysine and fibronectin, Artif Cells Blood Substit Immobil Biotechnol, vol. 34, no. 1, pp. 1125, 2006.
S. VandeVondele, J. Voros, and J. A. Hubbell, RGD-Grafted poly-l-lysine-graft-(polyethylene glycol)
copolymers block non-specific protein adsorption while promoting cell adhesion, Biotechnol. Bioeng., vol. 82,
no. 7, pp. 784790, 2003.
A. Agarwal, J. A. Goss, A. Cho, M. L. McCain, and K. K. Parker, Microfluidic heart on a chip for higher
throughput pharmacological studies, Lab on a Chip, vol. 13, no. 18, pp. 35993608, 2013.
K. Yum, S. G. Hong, K. E. Healy, and L. P. Lee, Physiologically relevant organs on chips, Biotechnology
Journal, vol. 9, no. 1, pp. 1627, Jan. 2014.
A. Natarajan, M. Stancescu, V. Dhir, C. Armstrong, F. Sommerhage, J. J. Hickman, and P. Molnar,
Patterned cardiomyocytes on microelectrode arrays as a functional, high information content drug screening
platform, Biomaterials, vol. 32, no. 18, pp. 42674274, Jun. 2011.
J. S. Leyton-Mange, R. W. Mills, V. S. Macri, M. Y. Jang, F. N. Butte, P. T. Ellinor, and D. J. Milan, Rapid
Cellular Phenotyping of Human Pluripotent Stem Cell-Derived Cardiomyocytes using a Genetically Encoded
Fluorescent Voltage Sensor, Stem Cell Reports, vol. 2, no. 2, pp. 163170, 2014.
A. Tourovskaia, M. Fauver, G. Kramer, S. Simonson, and T. Neumann, Tissue-engineered
microenvironment systems for modeling human vasculature., Exp. Biol. Med. (Maywood), vol. 239, no. 9, pp.
12641271, Sep. 2014.
K. M. French, A. V. Boopathy, J. A. DeQuach, L. Chingozha, H. Lu, K. L. Christman, and M. E. Davis, A
naturally derived cardiac extracellular matrix enhances cardiac progenitor cell behavior in vitro, Acta
Biomaterialia, vol. 8, no. 12, pp. 43574364, Dec. 2012.
S. F. Badylak, The extracellular matrix as a biologic scaffold material, Biomaterials, vol. 28, no. 25, pp.
35873593, Sep. 2007.

Appendix 1: Request for Proposal (RFP)

4/6/2015
Project title: A microphysiological system of the human heart for drug efficacy and toxicity testing
This proposal responds to a request from Dr. Deok-Ho Kims Research Group (The Kim Lab). The Kim Lab
seeks a solution to the lack of sufficiently predictive and cost-effective preclinical in vitro drug screens for
evaluating the arrhythmogenic potential of drug candidates. Specifically, The Kim Lab requests the
development of a biomimetic model capable of recapitulating functional attributes of native human cardiac
tissue. To date, no commercially available model exists that adequately replicates the in vivo myocardial niche
or human myocardial responses to chemical stimuli. The proposed project aims to develop a model of the
myocardium based on existing commercially available microfluidic technology to enable the attainment of more
predictive drug toxicity data prior to clinical trials.
A microfluidic in vitro model of the human heart with the following main attributes will be developed: (1)
a tissue-engineered myocardial cord consisting of human cardiomyocytes arranged into a physiologically
relevant architecture; (2) an extracellular matrix (ECM) that resembles the specific micro-environment of the
heart; (3) a genetically encoded calcium indicator (GCaMP3 expressed by RUES2 Cardiomyocytes) providing
real-time evaluation of tissue electrophysiological function; (4) microfluidic flow providing shear stress to the
engineered tissue, as well as effective drug delivery and removal; (5) tightly-controlled physical and chemical
conditions; (6) a mass-produced, disposable fluidic device that can be adapted for use in existing high-content
analysis as well as multi-organ platforms.
The proposed project will be divided into two main tasks:
(1) Define chip features, architecture of the microenvironment, ECM composition, and cell-culture
protocols. This first task is focused on adapting the current design of a commercially available chip
(Nortis) to meet the functional and read-out requirements of pharmaceutical development protocols.
This includes: optimizing the chip design, determining those elements of the cardiac microenvironment
that influence cardiac function, such as composition of the ECM, cell architecture, and physicochemical
factors. Consistency in the initial seeding of cardiac cells is crucial for achieving repeatable
electrophysiological data. The spatial distribution and maturation of the seeded cells will be evaluated
with standard fluorescent imaging tools.
(2) Establish protocols for assessing cardiac function in the chip (baseline and drug response).
This second task is designed to develop metrics for assessing cardiac function within the newly
developed chip. These metrics will be used to measure cardiac baseline performance as well as
responses to potentially cardio-active compounds such as cisapride (a human ether--go-go-related
gene (hERG) channel blocker), digoxin (Na+/K+-pump blocker), or camptothecin (anticancer agent) or
others which have been interrogated in the literature with respect to cardiac side effects. This will be
achieved through assessment of changes in calcium transients using a GCaMP3 reporter molecule
before, during, and after drug perfusion.

Page 13

Appendix 2: Concept Sheet

4/21/2015

BIOENGINEERING SOLUTIONS TO REAL WORLD PROBLEMS

A microphysiological system of the human heart for drug efficacy
and toxicity testing
Clinical need.

Cardiovascular disease is the leading cause of global mortality, killing

approximately 600,000 people each year in the United States alone - roughly 25% of all annual
US deaths (1). Despite the need for more advanced treatments, extant drug testing and approval
mechanisms are slow, expensive, and perhaps most importantly ineffective with respect to the
results they purport to produce. Significant findings concerning adverse drug effects, particularly
with respect to cardiac toxicity, have occurred in late stage clinical studies and even during postmarketing stages of drug commercialization. Given an estimated total pre-approval cost of $802
million, the cost of late-stage drug failure during development and screening can be on the order
of tens of millions of dollars (2). In the US, of the 95 compounds withdrawn from US drug
markets in the 40 years preceding 2009, 19 percent were withdrawn for cardiovascular safety
reasons, including 12 percent withdrawn specifically due to proarrhythmia (3). There is a pressing
urgency for biomedical research to develop more predictive ways of screening new drugs for
cardiotoxic effects.

Bioengineering solution.

With the intention of applying our knowledge and

techniques concerning the advanced maturation of engineered cardiac tissue, we now propose the
development of an in vitro microfluidic model of the human myocardium that overcomes many of
the limitations of current cardiac tissue mimics. Our model will employ tissue-engineered 3D
architecture to recapitulate the physiological anisotropy of native cardiac tissue, enabling cardiac
cell elongation, formation of multi-cell constructs of confluent tissue, and synchronous,
directional contractile activity. The use of multi-cell tissue constructs in our model will allow for
the formation of intercellular connections and mechanical organization and elongation,
characteristics that are essential to the maturation of cardiac tissue, and which are absent in extant
single-cell heart-on-a-chip models.

Current status and results.

Contemporary drug screening practices and

methodologies rely heavily on animal testing. Nonhuman animal tests are known to be extremely
poor proxies for determining drug effects in humans. The FDA reported in 2004 that 92 percent of
drugs that pass animal trials are later rejected when screened in humans. Broadly, in vivo
screening of drugs in the animal model is expensive, slow, fails to imitate human physiology, and
is often incapable of recapitulating human disease states.
Our research group has published extensively on the development of human stem cellderived cardiomyocyte tissue constructs with highly physiologic properties that very nearly
recapitulate cardiac tissue function in vivo (4-7). Preliminary data suggest that human
cardiomyocytes cultured in decellularized porcine cardiac ECM develop functionally mature cell
sheets as indicated by structural anisotropy, alignment and elongation, and contractile activity.
Co-injection of cardiomyocytes and dECM into the device lumen may promote more advanced
maturation, and consequently may lead to a more physiologic myocardial model. Additionally,
our research group has developed of a stably transfected GCaMP3 reporter expressing line of
human stem cell-derived cardiomyocytes. GCaMP3 is a genetically encoded calcium indicator,
which enables real-time optical observation of calcium signaling in living tissue constructs, a
practical proxy for the analysis of tissue function and contractile activity (8-10). These results
have been integrated with some proof-of-concept success into early stage prototypes of our
microfluidic model.

References: 1. Murphy SL, Xu JQ, Kochanek KD. Deaths: final data for 2010. National vital statistics reports. 2013;61(4):1118. 2.

Drug-induced
arrhythmia is one of the
most common causes of
drug development
failure and withdrawal
from market.
Navarrete, E. G., Liang, P., Lan,
F., Sanchez-Freire, V., Simmons,
C., Gong, T., et al. (2013).
Screening Drug-Induced
Arrhythmia Events Using Human
Induced Pluripotent Stem CellDerived Cardiomyocytes and LowImpedance Microelectrode Arrays.
Circulation , 128(11), S3U51.
http://doi.org/10.1161/CIRCULA
TIONAHA.112.000570

Personnel
Elliot Fisher
Mentor: Dr. Alec Smith

PI: Dr. Deok-Ho Kim

Concept Sheet

DiMasi JA, Hansen RW, Grabowski HG. The price of

innovation: new estimates of drug development costs. J Health Econ. 2003 Mar;22(2):15185. 3. Piccini JP, Whellan DJ, Berridge BR, Finkle JK, Pettit SD, Stockbridge N, et al. Current challenges in the
evaluation of cardiac safety during drug development: Translational medicine meets the Critical Path Initiative. American Heart Journal. 2009 Sep;158(3):31726. 4. Kim D-H, Kim P, Suh KY, Choi SK, Lee
SH, Kim B. Modulation of Adhesion and Growth of Cardiac Myocytes by Surface Nanotopography. 2005 IEEE Engineering in Medicine and Biology 27th Annual Conference. IEEE; 2006. 4 p. 5. Kim D-H,
Kshitiz, Smith RR, Kim P, Ahn EH, Kim HN, et al. Nanopatterned cardiac cell patches promote stem cell niche formation and myocardial regeneration. Integr Biol. The Royal Society of Chemistry;
2012;4(9):101933. 6. Kim D-H, Lipke EA, Kim P, Cheong R, Thompson S, Delannoy M, et al. Nanoscale cues regulate the structure and function of macroscopic cardiac tissue constructs. Proc Natl Acad Sci
USA. National Acad Sciences; 2010 Jan 12;107(2):56570. 7. Kim HN, Jiao A, Hwang NS, Kim MS, Kang D-H, Kim D-H, et al. Nanotopography-guided tissue engineering and regenerative medicine.
Advanced Drug Delivery Reviews. 2013 Apr;65(4):53658. 8. Shiba Y, Filice D, Fernandes S, Minami E, Dupras SK, Van Biber B, et al. Electrical Integration of Human Embryonic Stem Cell-Derived
Cardiomyocytes in a Guinea Pig Chronic Infarct Model. J Cardiovasc Pharmacol Ther. SAGE Publications; 2014 Jul;19(4):36881. 9. Chong JJH, Yang X, Don CW, Minami E, Liu Y-W, Weyers JJ, et al.
Human embryonic-stem-cell-derived cardiomyocytes regenerate non-human primate hearts. Nature. 2014 Jun 12;510(7504):2737. 10. Zhu W-Z, Filice D, Palpant NJ, Laflamme MA. Methods for assessing
the electromechanical integration of human pluripotent stem cell-derived cardiomyocyte grafts. Methods Mol Biol. New York, NY: Springer New York; 2014;1181(Chapter 20):22947.

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