With cardiovascular diseases representing a leading cause of death worldwide, several
biological aspects of heart physiology and pathology remain open and therapeutic needs
widely unmet. The attention of biomedical research has been directed toward the development
and exploitation of meaningful in vitro models of the myocardium, in both health and
disease. Indeed, an increasing need has risen for advanced platforms able to investigate
heart pathological mechanisms or development of drug treatments with low impact on
cardiac function.
An ideal in vitro heart model should be capable of tailoring environmental cues provided
to human cells or tissues (biochemical signals, physical stimulations) while also
providing means to analyze functional readouts (e.g., contractility of the tissue,
electrical activity). These specifics are the key to conduct both basic research investigations
(e.g., cellular mechanisms of cardiac pathologies) and translational studies (effects
of candidate drug compounds on heart functions). A class of culture platforms recently
acknowledged as prospective to meet these needs and advance these fields are organs-on-chips.
Exploiting photo and soft-lithography microfabrication techniques, organs-on-chip
provide microstructured arrangements of cells recapitulating key organ/tissue functions
and supplied by microfluidic channels [1]. The relatively low volume of fluids required
to culture cells within these devices makes it possible to focus the use of organs-on-chip
toward human cells (primary cells or derived from pluripotent stem cells), thus stepping
away from animal sources. These concepts have been applied to heart-derived cells
in order to more finely recapitulate the cardiac environment in vitro within so-called
heart-on-chips. To date, this rapidly growing field accounts for several reports describing
heart-on-chip designs [2], which in some instances were validated for carrying out
relevant drug screening studies [3]. Heart-on-chip models therefore hold concrete
potential for advancing the field of cell culture platform. However, they still require
improvements and directions in order to attain meaningful biological models for robustly
serving pharmaceutical screenings or basic research. We here comment on existing heart-on-chip
microfluidic devices, with an outlook on unmet requirements and future directions
for rational technological development.
Controlling physical & chemical environments
An inherent advantage of microfabricated organs-on-chip is the ability of applying
tailored physical and/or biochemical cues to cultured cells, in an attempt to more
closely mimic the cardiac cellular microenvironment. A prominent physical stimulus
in the heart is the cyclic mechanical loading caused by tissue contractility. The
mechanical microenvironment is known to have major effects on cardiac cells and has
been shown to drive cellular fate of several heart-related cell types [4].
A substantial number of investigations regarding mechanical loading platforms and
biological cellular responses have been carried out by means of microfluidic systems.
Flexible substrates allow to culture cellular monolayers and on-chip pneumatic actuation
systems typically provide pressure or vacuum-driven substrate stretching. This class
of microsystems led to significant developments in the exploration of cardiac cells
mechano-responses: embryonic stem cells were shown to exhibit a preferential cardiomyogenic
differentiation when subjected to cyclic mechanical stimuli [5]. In the field of fibrotic
response, human cardiac fibroblasts were shown to exhibit different responses according
to the intensity of mechanical stimuli applied [6,7]. Cardiac myocytes 3D constructs
exhibited synchronous contraction only under mechanical stimulation, with unorganized
patterns of contraction in the nonstimulated counterparts [8]. Of note, recent technical
developments include a design solution for coupling 3D hydrogel-based constructs with
cyclic mechanical stimulation in microfluidics [8], thus enabling the fabrication
of organs-on-chip that combine the physiological architecture of 3D ECM-based constructs
with the possibility of mechanically stimulate them.
Electrical stimulation is another key feature reproduced in vitro to better recapitulate
the in vivo cardiac environment. In the heart, cardiac cells are electrically coupled
and continuously subjected to electric currents. Biomimetic electric signals have
been demonstrated to enhance conductive and contractile properties of cardiomyocytes
and to influence stem cell fate [9]. Several microfluidic platforms have been developed
to provide cells with different electrical stimulation patterns, applied alone or
in concert with other stimulations (i.e., chemical, mechanical, topological). 2D flat
electrodes obtained through electrodeposition [10] or laser ablation [11] technologies
can be directly integrated in microfluidic platform surfaces, where cellular monolayers
are cultured. Alternatively, 3D electrodes can be formed by injecting conductive compounds
in a predesigned compartment of the platform [12] or can be achieved directly inserting
physical electrodes (platinum wires, carbon rods, stainless steel) in contact with
cell culture medium.
Electric fields applied on cardiomyocytes monolayers successfully provided appropriate
environmental cues to maintain cardiac phenotype and contractile function in vitro,
enhancing cellular elongation, cardiac maturation, beating performances and gap junction
organization [11]. In heart-on-chip systems housing 3D cell-laden hydrogels, electrical
stimulation allowed, for instance, human pluripotent stem cells [13] derived cardiomyocytes
to mature in a tissue-like constructs, enhancing construct organization and conduction
velocity.
Standard systems for cardiac functional readouts
While microenvironmental cues are key factors for aiding cardiac constructs maturation
and mimicking physiological or pathological states, another crucial aspect in designing
heart-on-chip devices is the inclusion of readout systems for assessing cardiac biological
functions. Contractility is the hallmark of heart function and contractile force quantification
is one of the most important functional characterizations of cardiac muscle. Over
the past decades, enormous efforts have been made to develop techniques that overcome
the challenges in assessing the contractile properties of cardiomyocytes. Huebsch et al. [14]
adopted a force transducer system technique to assess microheart muscle physiology.
This technique required harsh manipulations of the tissue: microheart muscles were
microdissected, removed from its original platform, hooked and immersed in a bath.
Although, often used two-point force assays measure force along a single axis, neglecting
multidirectional contraction exerted by cells whose cytoskeleton is unaligned (e.g., immature
cardiomyocytes).
Some appealing, noninvasive approaches for force measurement are based on small cantilever
deflections. In atomic force microscopy, a small cantilever gently touches a beating
cardiomyocyte and its vertical deflection tracks the beating force signal [15]. Nonetheless,
atomic force microscopy instrumentation has a low chance of being integrated in compact
systems. In a pivotal work, Tanaka et al. [16] demonstrated a cheaper solution to
measure single-cell contractile force, by using polydimethylsiloxane(PDMS)-based arrays
of micropillars that deflect according to the motion of attached cardiomyocytes. Their
strategy requires video analysis and mathematical estimations that depend on pillar
shape and on cardiomyocytes attachment.
More recently, progress has been made toward high-resolution 2D mapping of cell tractions
at cell surface. Traction force microscopy (TFM) has been widely used to characterize
single cardiomyocyte contractility [17], exploiting cardiomyocytes ability to wrinkle
an elastic substrate during contraction. TFM consists on tracking displacements of
nanobeads embedded on a hydrogel substrate. Knowing the mechanical characteristics
of the substrate and using mathematical estimations TFM allows to quantify the traction
stress exerted by contracting cells. The main advantage of TFM is that it is noninvasive,
nonterminal and it only requires basic equipment to assess cardiomyocytes contractile
performance. Video microscopy simplifies TFM by removing the need of beads embedded
in the substrate: indeed, estimated strain fields of complex cell distortions rely
on standalone high-quality image acquisition [18].
Another important challenge related to cardiac model generation has been the investigation
of electrophysiological characteristics of cultured cells. The ability of quantitatively
assessing functional parameters such as spontaneous beating frequency, depolarization-repolarization
patterns and ionic currents magnitude is particularly appealing to drug screening
and regenerative medicine applications. Different techniques are available to perform
measurements of single cell electrophysiological activity.
To monitor and directly measure extracellular potential of interconnected cellular
monolayers in vitro, microarrays of electrode (MEAs) have been developed [19]. MEA
technology consists of arrays of microelectrodes (made of indium tin oxide or titanium)
patterned onto a glass surface that permits simultaneous stimulation and recording
of extracellular field potentials generated by cell action potentials. Different electrode
patterns (from 8 × 8 up to 16 × 16 grid) can be achieved, allowing for recording signal
with high spatial and temporal resolution. Conversely to patch clamp, the most prominent
advantage of MEAs relies on the possibility to achieve long-term and noninvasive monitoring
of large cell population. For this reason, MEAs have been widely exploited to conduct
safety pharmacology studies directly on monolayers of human-induced pluripotent stem
cell derived cardiomyocytes, monitoring real-time changes in cardiac action potential
duration after drug administration [20]. In order to combine the long-term, noninvasive
MEA measurements with the possibility to record intracellular action potentials, protruding
gold mushroom-shaped microelectrodes were also developed [21]. This system allows
the repeated intracellular recording of cardiomyocyte action potential after an electroporation
pulse that temporarily disrupts the cell membrane.
All the above-mentioned technologies are efficient for single cells or interconnected
cell monolayer electrical measurements and were successfully integrated in standard
2D culture platforms. However there is an urgent need to integrate electrical measurement
systems within advanced microfluidic platforms, where chemical and physical stimulations
are tailored to generate in vitro complex cardiac models.
Integrating functional readouts in heart-on-chips
Perspective heart-on-a-chip systems integrate strategies that allow quantitative functional
analysis of cardiac tissue parameters. The major advantage of standalone microfluidic
devices is that they do not require complicated and potentially disruptive tissue
manipulations, while continuously assessing cardiac functional readouts along the
whole culture period.
In terms of contractility, muscular thin films (MTF) are 2D bilaminate engineered
cultures that recapitulate tissue-scale cardiac contractile behavior. Cardiomyocytes
were cultured on deformable thin films that bend in response to the systolic stress
generated. Recently, Lind et al. [22] upgraded the manufacturing procedure concept
and used multimaterial 3D printing technique to fabricate an MTF-inspired heart-on-a-chip.
Fully automated acquisition of twitch data was made possible by integrating soft strain
gauge sensors within the device. Despite the high automation achieved, MTF strategy
does not recapitulate the 3D complex native heart tissue organization.
In microfluidic-based 3D cardiac tissue model configurations, the main data acquisition
obstacles are related to highly dense and tightly packed cell configurations that
make individual cell analysis challenging. Hence, systems aim at determining the overall
mechanical properties of the bulk cardiac tissue. For instance, video-based methods
were employed by Mathur et al. [23] to analyze time-averaged beating motion and beating
kinetics of cardiac microphysiological systems. In general, force quantification of
the 3D cardiac constructs by video microscopy is likely to be affected by analysis
conditions. Tissue anchors to bulky walls of microdevice that withstand tissue contractions,
making moving edges insignificant or undetectable.
In terms of electrical activity recordings, only a few studies report the integration
of direct recording systems within heart-on-chip microfluidic platforms. Platinum
electroplated planar electrodes in combination with microfluidic networks were used
to measure extracellular potentials of single entrapped cardiomyocytes during spontaneous
contraction [24]. With a similar approach, patterned Ag/AgCl electrodes placed in
two separate microchannels connected by cells are exploited to both stimulate and
record cardiac action potential of single-cell or pairs of cardiomyocytes [25].
Microfluidic platforms integrating MEA surfaces have also been developed. Agarose
microstructures or PDMS-based culture chambers developed on top of MEAs allow the
precise control of spatial cell arrangement, enabling investigations of cardiac electrical
activity spatiotemporal development. This technique can be applied to record cardiomyocyte
field potential fluctuations in response to drug administration [26], to assess cardiac
modulation exerted by sympathetic nervous system [27] or to investigate conduction
velocity and cellular physiological functionality after an ischemic event, testing
the capabilities of different cell sources to form new gap junctions [28].
Noteworthy, none of the previously described methods (patch clamp or gold mushroom-shaped
microelectrodes) allowing for assessing information on intracellular electric potential
have yet been coupled to microfluidic platforms. Furthermore, none of the 3D in vitro
cardiac microfluidic models include electrical activity recording systems.
Conclusion
The number of organs-on-chip focused on heart physiology is continuously rising and
ever more complex microenvironments are tailored for heart cells growth and analysis.
The existing literature is widely focused on tailoring specific on-chip environmental
cues, particularly electromechanical stimuli, to generate cardiac biological models.
However, in order to fulfill the goal of stand-alone platforms for high-throughput
analyses of cardiac models performances in basic research or drug screening, a deeper
focus in the direction of integrating cardiac cells functional assessments is required.
With a successful integration of cardiac output measurement systems, the existing
heart-mimicking microfluidic organs-on-chips are expected to pave the way to a new
generation of in vitro testing capabilities.