SARS-CoV-2 pathogenesis in an angiotensin II-induced heart-on-a-chip disease model and extracellular vesicle screening.

Adverse cardiac outcomes in COVID-19 patients, particularly those with preexisting cardiac disease, motivate the development of human cell-based organ-on-a-chip models to recapitulate cardiac injury and dysfunction and for screening of cardioprotective therapeutics. Here, we developed a heart-on-a-chip model to study the pathogenesis of SARS-CoV-2 in healthy myocardium established from human induced pluripotent stem cell (iPSC)-derived cardiomyocytes and a cardiac dysfunction model, mimicking aspects of preexisting hypertensive disease induced by angiotensin II (Ang II). We recapitulated cytopathic features of SARS-CoV-2-induced cardiac damage, including progressively impaired contractile function and calcium handling, apoptosis, and sarcomere disarray. SARS-CoV-2 presence in Ang II-treated hearts-on-a-chip decreased contractile force with earlier onset of contractile dysfunction and profoundly enhanced inflammatory cytokines compared to SARS-CoV-2 alone. Toward the development of potential therapeutics, we evaluated the cardioprotective effects of extracellular vesicles (EVs) from human iPSC which alleviated the impairment of contractile force, decreased apoptosis, reduced the disruption of sarcomeric proteins, and enhanced beta-oxidation gene expression. Viral load was not affected by either Ang II or EV treatment. We identified MicroRNAs miR-20a-5p and miR-19a-3p as potential mediators of cardioprotective effects of these EVs.

Biofabrication strategies for cardiac tissue engineering.

Biofabrication technologies hold the potential to provide high-throughput, easy-to-operate, and cost-effective systems that recapitulate complexities of the native heart. The size of the fabricated model, printing resolution, biocompatibility, and ease-of-fabrication are some of the major parameters that can be improved to develop more sophisticated cardiac models. Here, we review recent cardiac engineering technologies ranging from microscaled organoids, millimeter-scaled heart-on-a-chip platforms, in vitro ventricle models sized to the fetal heart, larger cardiac patches seeded with billions of cells, and associated biofabrication technologies used to produce these models. Finally, advancements that facilitate model translation are discussed, such as their application as carriers for bioactive components and cells in vivo or their capability for drug testing and disease modeling in vitro.

Primitive macrophages enable long-term vascularization of human heart-on-a-chip platforms.

The intricate anatomical structure and high cellular density of the myocardium complicate the bioengineering of perfusable vascular networks within cardiac tissues. In vivo neonatal studies highlight the key role of resident cardiac macrophages in post-injury regeneration and angiogenesis. Here, we integrate human pluripotent stem-cell-derived primitive yolk-sac-like macrophages within vascularized heart-on-chip platforms. Macrophage incorporation profoundly impacted the functionality and perfusability of microvascularized cardiac tissues up to 2 weeks of culture. Macrophages mitigated tissue cytotoxicity and the release of cell-free mitochondrial DNA (mtDNA), while upregulating the secretion of pro-angiogenic, matrix remodeling, and cardioprotective cytokines. Bulk RNA sequencing (RNA-seq) revealed an upregulation of cardiac maturation and angiogenesis genes. Further, single-nuclei RNA sequencing (snRNA-seq) and secretome data suggest that macrophages may prime stromal cells for vascular development by inducing insulin like growth factor binding protein 7 (IGFBP7) and hepatocyte growth factor (HGF) expression. Our results underscore the vital role of primitive macrophages in the long-term vascularization of cardiac tissues, offering insights for therapy and advancing heart-on-a-chip technologies.

Heart-on-a-Chip Model of Epicardial-Myocardial Interaction in Ischemia Reperfusion Injury.

Epicardial cells (EPIs) form the outer layer of the heart and play an important role in development and disease. Current heart-on-a-chip platforms still do not fully mimic the native cardiac environment due to the absence of relevant cell types, such as EPIs. Here, using the Biowire II platform, engineered cardiac tissues with an epicardial outer layer and inner myocardial structure are constructed, and an image analysis approach is developed to track the EPI cell migration in a beating myocardial environment. Functional properties of EPI cardiac tissues improve over two weeks in culture. In conditions mimicking ischemia reperfusion injury (IRI), the EPI cardiac tissues experience less cell death and a lower impact on functional properties. EPI cell coverage is significantly reduced and more diffuse under normoxic conditions compared to the post-IRI conditions. Upon IRI, migration of EPI cells into the cardiac tissue interior is observed, with contributions to alpha smooth muscle actin positive cell population. Altogether, a novel heart-on-a-chip model is designed to incorporate EPIs through a formation process that mimics cardiac development, and this work demonstrates that EPI cardiac tissues respond to injury differently than epicardium-free controls, highlighting the importance of including EPIs in heart-on-a-chip constructs that aim to accurately mimic the cardiac environment.

Itaconate and citrate releasing polymer attenuates foreign body response in biofabricated cardiac patches.

Application of cardiac patches to the heart surface can be undertaken to provide support and facilitate regeneration of the damaged cardiac tissue following ischemic injury. Biomaterial composition is an important consideration in the design of cardiac patch materials as it governs host response to ultimately prevent the undesirable fibrotic response. Here, we investigate a novel patch material, poly (itaconate-co-citrate-co-octanediol) (PICO), in the context of cardiac implantation. Citric acid (CA) and itaconic acid (ITA), the molecular components of PICO, provided a level of protection for cardiac cells during ischemic reperfusion injury in vitro. Biofabricated PICO patches were shown to degrade in accelerated and hydrolytic conditions, with CA and ITA being released upon degradation. Furthermore, the host response to PICO patches after implantation on rat epicardium in vivo was explored and compared to two biocompatible cardiac patch materials, poly (octamethylene (anhydride) citrate) (POMaC) and poly (ethylene glycol) diacrylate (PEGDA). PICO patches resulted in less macrophage infiltration and lower foreign body giant cell reaction compared to the other materials, with corresponding reduction in smooth muscle actin-positive vessel infiltration into the implant region. Overall, this work demonstrates that PICO patches release CA and ITA upon degradation, both of which demonstrate cardioprotective effects on cardiac cells after ischemic injury, and that PICO patches generate a reduced inflammatory response upon implantation to the heart compared to other materials, signifying promise for use in cardiac patch applications.

Advances in cardiac tissue engineering and heart-on-a-chip.

Recent advances in both cardiac tissue engineering and hearts-on-a-chip are grounded in new biomaterial development as well as the employment of innovative fabrication techniques that enable precise control of the mechanical, electrical, and structural properties of the cardiac tissues being modelled. The elongated structure of cardiomyocytes requires tuning of substrate properties and application of biophysical stimuli to drive its mature phenotype. Landmark advances have already been achieved with induced pluripotent stem cell-derived cardiac patches that advanced to human testing. Heart-on-a-chip platforms are now commonly used by a number of pharmaceutical and biotechnology companies. Here, we provide an overview of cardiac physiology in order to better define the requirements for functional tissue recapitulation. We then discuss the biomaterials most commonly used in both cardiac tissue engineering and heart-on-a-chip, followed by the discussion of recent representative studies in both fields. We outline significant challenges common to both fields, specifically: scalable tissue fabrication and platform standardization, improving cellular fidelity through effective tissue vascularization, achieving adult tissue maturation, and ultimately developing cryopreservation protocols so that the tissues are available off the shelf.

High-Resolution Additive Manufacturing of a Biodegradable Elastomer with A Low-Cost LCD 3D Printer.

Artificial organs and organs-on-a-chip (OoC) are of great clinical and scientific interest and have recently been made by additive manufacturing, but depend on, and benefit from, biocompatible, biodegradable, and soft materials. Poly(octamethylene maleate (anhydride) citrate (POMaC) meets these criteria and has gained popularity, and as in principle, it can be photocured and is amenable to vat-photopolymerization (VP) 3D printing, but only low-resolution structures have been produced so far. Here, a VP-POMaC ink is introduced and 3D printing of 80 µm positive features and complex 3D structures is demonstrated using low-cost (≈US$300) liquid-crystal display (LCD) printers. The ink includes POMaC, a diluent and porogen additive to reduce viscosity within the range of VP, and a crosslinker to speed up reaction kinetics. The mechanical properties of the cured ink are tuned to match the elastic moduli of different tissues simply by varying the porogen concentration. The biocompatibility is assessed by cell culture which yielded 80% viability and the potential for tissue engineering illustrated with a 3D-printed gyroid seeded with cells. VP-POMaC and low-cost LCD printers make the additive manufacturing of high resolution, elastomeric, and biodegradable constructs widely accessible, paving the way for a myriad of applications in tissue engineering and 3D cell culture as demonstrated here, and possibly in OoC, implants, wearables, and soft robotics.

Elastomeric Polyesters in Cardiovascular Tissue Engineering and Organs-on-a-Chip.

Cardiovascular tissue constructs provide unique design requirements due to their functional responses to substrate mechanical properties and cyclic stretching behavior of cardiac tissue that requires the use of durable elastic materials. Given the diversity of polyester synthesis approaches, an opportunity exists to develop a new class of biocompatible, elastic, and immunomodulatory cardiovascular polymers. Furthermore, elastomeric polyester materials have the capability to provide tailored biomechanical synergy with native tissue and hence reduce inflammatory response in vivo and better support tissue maturation in vitro. In this review, we highlight underlying chemistry and design strategies of polyester elastomers optimized for cardiac tissue scaffolds. The major advantages of these materials such as their tunable elasticity, desirable biodegradation, and potential for incorporation of bioactive compounds are further expanded. Unique fabrication methods using polyester materials such as micromolding, 3D stamping, electrospinning, laser ablation, and 3D printing are discussed. Moreover, applications of these biomaterials in cardiovascular organ-on-a-chip devices and patches are analyzed. Finally, we outline unaddressed challenges in the field that need further study to enable the impactful translation of soft polyesters to clinical applications.

Flexible 3D printed microwires and 3D microelectrodes for heart-on-a-chip engineering.

We developed a heart-on-a-chip platform that integrates highly flexible, vertical, 3D micropillar electrodes for electrophysiological recording and elastic microwires for the tissue's contractile force assessment. The high aspect ratio microelectrodes were 3D-printed into the device using a conductive polymer, poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate) (PEDOT:PSS). A pair of flexible, quantum dots/thermoplastic elastomer nanocomposite microwires were 3D printed to anchor the tissue and enable continuous contractile force assessment. The 3D microelectrodes and flexible microwires enabled unobstructed human iPSC-based cardiac tissue formation and contraction, suspended above the device surface, under both spontaneous beating and upon pacing with a separate set of integrated carbon electrodes. Recording of extracellular field potentials using the PEDOT:PSS micropillars was demonstrated with and without epinephrine as a model drug, non-invasively, along within situmonitoring of tissue contractile properties and calcium transients. Uniquely, the platform provides integrated profiling of electrical and contractile tissue properties, which is critical for proper evaluation of complex, mechanically and electrically active tissues, such as the heart muscle under both physiological and pathological conditions.

Intersection of stem cell biology and engineering towards next generation in vitro models of human fibrosis.

Human fibrotic diseases constitute a major health problem worldwide. Fibrosis involves significant etiological heterogeneity and encompasses a wide spectrum of diseases affecting various organs. To date, many fibrosis targeted therapeutic agents failed due to inadequate efficacy and poor prognosis. In order to dissect disease mechanisms and develop therapeutic solutions for fibrosis patients, in vitro disease models have gone a long way in terms of platform development. The introduction of engineered organ-on-a-chip platforms has brought a revolutionary dimension to the current fibrosis studies and discovery of anti-fibrotic therapeutics. Advances in human induced pluripotent stem cells and tissue engineering technologies are enabling significant progress in this field. Some of the most recent breakthroughs and emerging challenges are discussed, with an emphasis on engineering strategies for platform design, development, and application of machine learning on these models for anti-fibrotic drug discovery. In this review, we discuss engineered designs to model fibrosis and how biosensor and machine learning technologies combine to facilitate mechanistic studies of fibrosis and pre-clinical drug testing.

Toward Hierarchical Assembly of Aligned Cell Sheets into a Conical Cardiac Ventricle Using Microfabricated Elastomers.

Despite current efforts in organ-on-chip engineering to construct miniature cardiac models, they often lack some physiological aspects of the heart, including fiber orientation. This motivates the development of bioartificial left ventricle models that mimic the myofiber orientation of the native ventricle. Herein, an approach relying on microfabricated elastomers that enables hierarchical assembly of 2D aligned cell sheets into a functional conical cardiac ventricle is described. Soft lithography and injection molding techniques are used to fabricate micro-grooves on an elastomeric polymer scaffold with three different orientations ranging from -60° to +60°, each on a separate trapezoidal construct. The width of the micro-grooves is optimized to direct the majority of cells along the groove direction and while periodic breaks are used to promote cell-cell contact. The scaffold is wrapped around a central mandrel to obtain a conical-shaped left ventricle model inspired by the size of a human left ventricle 19 weeks post-gestation. Rectangular micro-scale holes are incorporated to alleviate oxygen diffusional limitations within the 3D scaffold. Cardiomyocytes within the 3D left ventricle constructs showed high viability in all layers after 7 days of cultivation. The hierarchically assembled left ventricle also provided functional readouts such as calcium transients and ejection fraction.

Engineering Models of the Heart Left Ventricle.

Despite capturing the imagination of scientists for decades, the goal of creating an artificial heart for transplantation proved to be significantly more challenging than initially anticipated. Toward this goal, recent ground-breaking studies demonstrate the development of functional left ventricular (LV) models. LV models are artificially constructed 3D chambers that are capable of containing liquid within the engineered cavity and exhibit the functionality of native LV including contraction, ejection of fluid, and electrical impulse propagation. Various hydrogels and polymers have been used in manufacturing of LV models, relying on techniques such as electrospinning, bioprinting, casting, and molding. Most studies scaled down the models based on the dimensions of the human or rat ventricle. Initially, neonatal rat cardiomyocytes were the cell type of choice for construction the LV models. Yet, as the stem cell biology field advanced, recent studies focused on the use of cardiomyocytes derived from human induced pluripotent stem cells. In this review, we first describe the physiological characteristics of the human heart, to establish the parameter space for modeling. We then elaborate on current advances in the field and compare recently developed LV models among themselves and with the native human left ventricle. Fabrication methods, cell types, biomaterials, functional properties, and disease modeling capability are some of the major parameters that have distinguished these models. We also highlight some of the current challenges in this field, such as vascularization, cell composition and fidelity, and discuss potential solutions to overcome them.

Beyond Polydimethylsiloxane: Alternative Materials for Fabrication of Organ-on-a-Chip Devices and Microphysiological Systems.

Polydimethylsiloxane (PDMS) is the predominant material used for organ-on-a-chip devices and microphysiological systems (MPSs) due to its ease-of-use, elasticity, optical transparency, and inexpensive microfabrication. However, the absorption of small hydrophobic molecules by PDMS and the limited capacity for high-throughput manufacturing of PDMS-laden devices severely limit the application of these systems in personalized medicine, drug discovery, in vitro pharmacokinetic/pharmacodynamic (PK/PD) modeling, and the investigation of cellular responses to drugs. Consequently, the relatively young field of organ-on-a-chip devices and MPSs is gradually beginning to make the transition to alternative, nonabsorptive materials for these crucial applications. This review examines some of the first steps that have been made in the development of organ-on-a-chip devices and MPSs composed of such alternative materials, including elastomers, hydrogels, thermoplastic polymers, and inorganic materials. It also provides an outlook on where PDMS-alternative devices are trending and the obstacles that must be overcome in the development of versatile devices based on alternative materials to PDMS.