A Platform for Generation of Chamber-Specific Cardiac Tissues and Disease Modeling.

Tissue engineering using cardiomyocytes derived from human pluripotent stem cells holds a promise to revolutionize drug discovery, but only if limitations related to cardiac chamber specification and platform versatility can be overcome. We describe here a scalable tissue-cultivation platform that is cell source agnostic and enables drug testing under electrical pacing. The plastic platform enabled on-line noninvasive recording of passive tension, active force, contractile dynamics, and Ca2+ transients, as well as endpoint assessments of action potentials and conduction velocity. By combining directed cell differentiation with electrical field conditioning, we engineered electrophysiologically distinct atrial and ventricular tissues with chamber-specific drug responses and gene expression. We report, for the first time, engineering of heteropolar cardiac tissues containing distinct atrial and ventricular ends, and we demonstrate their spatially confined responses to serotonin and ranolazine. Uniquely, electrical conditioning for up to 8 months enabled modeling of polygenic left ventricular hypertrophy starting from patient cells.

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.

An organ-on-a-chip model for pre-clinical drug evaluation in progressive non-genetic cardiomyopathy.

Angiotensin II (Ang II) presents a critical mediator in various pathological conditions such as non-genetic cardiomyopathy. Osmotic pump infusion in rodents is a commonly used approach to model cardiomyopathy associated with Ang II. However, profound differences in electrophysiology and pharmacokinetics between rodent and human cardiomyocytes may limit predictability of animal-based experiments. This study investigates the application of an Organ-on-a-chip (OOC) system in modeling Ang II-induced progressive cardiomyopathy. The disease model is constructed to recapitulate myocardial response to Ang II in a temporal manner. The long-term tissue cultivation and non-invasive functional readouts enable monitoring of both acute and chronic cardiac responses to Ang II stimulation. Along with mapping of cytokine secretion and proteomic profiles, this model presents an opportunity to quantitatively measure the dynamic pathological changes that could not be otherwise identified in animals. Further, we present this model as a testbed to evaluate compounds that target Ang II-induced cardiac remodeling. Through assessing the effects of losartan, relaxin, and saracatinib, the drug screening data implicated multifaceted cardioprotective effects of relaxin in restoring contractile function and reducing fibrotic remodeling. Overall, this study provides a controllable platform where cardiac activities can be explicitly observed and tested over the pathological process. The facile and high-content screening can facilitate the evaluation of potential drug candidates in the pre-clinical stage.

Biophysical stimulation for in vitro engineering of functional cardiac tissues.

Engineering functional cardiac tissues remains an ongoing significant challenge due to the complexity of the native environment. However, our growing understanding of key parameters of the in vivo cardiac microenvironment and our ability to replicate those parameters in vitro are resulting in the development of increasingly sophisticated models of engineered cardiac tissues (ECT). This review examines some of the most relevant parameters that may be applied in culture leading to higher fidelity cardiac tissue models. These include the biochemical composition of culture media and cardiac lineage specification, co-culture conditions, electrical and mechanical stimulation, and the application of hydrogels, various biomaterials, and scaffolds. The review will also summarize some of the recent functional human tissue models that have been developed for in vivo and in vitro applications. Ultimately, the creation of sophisticated ECT that replicate native structure and function will be instrumental in advancing cell-based therapeutics and in providing advanced models for drug discovery and testing.

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.

Automated fabrication of a scalable heart-on-a-chip device by 3D printing of thermoplastic elastomer nanocomposite and hot embossing.

The successful translation of organ-on-a-chip devices requires the development of an automated workflow for device fabrication, which is challenged by the need for precise deposition of multiple classes of materials in micro-meter scaled configurations. Many current heart-on-a-chip devices are produced manually, requiring the expertise and dexterity of skilled operators. Here, we devised an automated and scalable fabrication method to engineer a Biowire II multiwell platform to generate human iPSC-derived cardiac tissues. This high-throughput heart-on-a-chip platform incorporated fluorescent nanocomposite microwires as force sensors, produced from quantum dots and thermoplastic elastomer, and 3D printed on top of a polystyrene tissue culture base patterned by hot embossing. An array of built-in carbon electrodes was embedded in a single step into the base, flanking the microwells on both sides. The facile and rapid 3D printing approach efficiently and seamlessly scaled up the Biowire II system from an 8-well chip to a 24-well and a 96-well format, resulting in an increase of platform fabrication efficiency by 17,5000-69,000% per well. The device's compatibility with long-term electrical stimulation in each well facilitated the targeted generation of mature human iPSC-derived cardiac tissues, evident through a positive force-frequency relationship, post-rest potentiation, and well-aligned sarcomeric apparatus. This system's ease of use and its capacity to gauge drug responses in matured cardiac tissue make it a powerful and reliable platform for rapid preclinical drug screening and development.

Zeolitic imidazolate frameworks activate endosomal Toll-like receptors and potentiate immunogenicity of SARS-CoV-2 spike protein trimer.

Nanomaterials offer unique opportunities to engineer immunomodulatory activity. In this work, we report the Toll-like receptor agonist activity of a nanoscale adjuvant zeolitic imidazolate framework-8 (ZIF-8). The accumulation of ZIF-8 in endosomes and the pH-responsive release of its subunits enable selective engagement with endosomal Toll-like receptors, minimizing the risk of off-target activation. The intrinsic adjuvant properties of ZIF-8, along with the efficient delivery and biomimetic presentation of a severe acute respiratory syndrome coronavirus 2 spike protein receptor-binding domain trimer, primed rapid humoral and cell-mediated immunity in a dose-sparing manner. Our study offers insights for next-generation adjuvants that can potentially impact future vaccine development.

Recent advances in nano- and micro-scale carrier systems for controlled delivery of vaccines.

Vaccines provide substantial safety against infectious diseases, saving millions of lives each year. The recent COVID-19 pandemic highlighted the importance of vaccination in providing mass-scale immunization against outbreaks. However, the delivery of vaccines imposes a unique set of challenges due to their large molecular size and low room temperature stability. Advanced biomaterials and delivery systems such as nano- and mciro-scale carriers are becoming critical components for successful vaccine development. In this review, we provide an updated overview of recent advances in the development of nano- and micro-scale carriers for controlled delivery of vaccines, focusing on carriers compatible with nucleic acid-based vaccines and therapeutics that emerged amid the recent pandemic. We start by detailing nano-scale delivery systems, focusing on nanoparticles, then move on to microscale systems including hydrogels, microparticles, and 3D printed microneedle patches. Additionally, we delve into emerging methods that move beyond traditional needle-based applications utilizing innovative delivery systems. Future challenges for clinical translation and manufacturing in this rapidly advancing field are also discussed.

Organs-on-a-chip: a union of tissue engineering and microfabrication.

We review the emergence of the new field of organ-on-a-chip (OOAC) engineering, from the parent fields of tissue engineering and microfluidics. We place into perspective the tools and capabilities brought into the OOAC field by early tissue engineers and microfluidics experts. Liver-on-a-chip and heart-on-a-chip are used as two case studies of systems that heavily relied on tissue engineering techniques and that were amongst the first OOAC models to be implemented, motivated by the need to better assess toxicity to human tissues in preclinical drug development. We review current challenges in OOAC that often stem from the same challenges in the parent fields, such as stable vascularization and drug absorption.

Maturation of iPSC-derived cardiomyocytes in a heart-on-a-chip device enables modeling of dilated cardiomyopathy caused by R222Q-SCN5A mutation.

To better understand sodium channel (SCN5A)-related cardiomyopathies, we generated ventricular cardiomyocytes from induced pluripotent stem cells obtained from a dilated cardiomyopathy patient harbouring the R222Q mutation, which is only expressed in adult SCN5A isoforms. Because the adult SCN5A isoform was poorly expressed, without functional differences between R222Q and control in both embryoid bodies and cell sheet preparations (cultured for 29-35 days), we created heart-on-a-chip biowires which promote myocardial maturation. Indeed, biowires expressed primarily adult SCN5A with R222Q preparations displaying (arrhythmogenic) short action potentials, altered Na+ channel biophysical properties and lower contractility compared to corrected controls. Comprehensive RNA sequencing revealed differential gene regulation between R222Q and control biowires in cellular pathways related to sarcoplasmic reticulum and dystroglycan complex as well as biological processes related to calcium ion regulation and action potential. Additionally, R222Q biowires had marked reductions in actin expression accompanied by profound sarcoplasmic disarray, without differences in cell composition (fibroblast, endothelial cells, and cardiomyocytes) compared to corrected biowires. In conclusion, we demonstrate that in addition to altering cardiac electrophysiology and Na+ current, the R222Q mutation also causes profound sarcomere disruptions and mechanical destabilization. Possible mechanisms for these observations are discussed.

Design and Fabrication of Biological Wires for Cardiac Fibrosis Disease Modeling.

Extensive progress has been made in developing engineered models for elucidating human cardiac disease. Cardiac fibrosis is often associated with all forms of cardiac disease and has a direct deleterious effect on cardiac function. As currently there is no effective therapeutic strategy specifically designed to target fibrosis, in vitro diagnostic platforms for drug testing have generated significant interest. In this context, we have developed an innovative approach to generate human cardiac fibrotic tissues on Biowire II platform and established a compound screening system. The disease model is constructed to recapitulate contractile, biomechanical, and electrophysiological complexities of fibrotic myocardium. Additionally, an integrated model with fibrotic and healthy cardiac tissues coupled together can be created to mimic focal fibrosis. The methods for constructing the Biowire fibrotic model will be described here.

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.

A well plate-based multiplexed platform for incorporation of organoids into an organ-on-a-chip system with a perfusable vasculature.

Owing to their high spatiotemporal precision and adaptability to different host cells, organ-on-a-chip systems are showing great promise in drug discovery, developmental biology studies and disease modeling. However, many current micro-engineered biomimetic systems are limited in technological application because of culture media mixing that does not allow direct incorporation of techniques from stem cell biology, such as organoids. Here, we describe a detailed alternative method to cultivate millimeter-scale functional vascularized tissues on a biofabricated platform, termed 'integrated vasculature for assessing dynamic events', that enables facile incorporation of organoid technology. Utilizing the 3D stamping technique with a synthetic polymeric elastomer, a scaffold termed 'AngioTube' is generated with a central microchannel that has the mechanical stability to support a perfusable vascular system and the self-assembly of various parenchymal tissues. We demonstrate an increase in user familiarity and content analysis by situating the scaffold on a footprint of a 96-well plate. Uniquely, the platform can be used for facile connection of two or more tissue compartments in series through a common vasculature. Built-in micropores enable the studies of cell invasion involved in both angiogenesis and metastasis. We describe how this protocol can be applied to create both vascularized cardiac and hepatic tissues, metastatic breast cancer tissue and personalized pancreatic cancer tissue through incorporation of patient-derived organoids. Platform assembly to populating the scaffold with cells of interest into perfusable functional vascularized tissue will require 12-14 d and an additional 4 d if pre-polymer and master molds are needed.

Towards chamber specific heart-on-a-chip for drug testing applications.

Modeling of human organs has long been a task for scientists in order to lower the costs of therapeutic development and understand the pathological onset of human disease. For decades, despite marked differences in genetics and etiology, animal models remained the norm for drug discovery and disease modeling. Innovative biofabrication techniques have facilitated the development of organ-on-a-chip technology that has great potential to complement conventional animal models. However, human organ as a whole, more specifically the human heart, is difficult to regenerate in vitro, in terms of its chamber specific orientation and its electrical functional complexity. Recent progress with the development of induced pluripotent stem cell differentiation protocols, made recapitulating the complexity of the human heart possible through the generation of cells representative of atrial & ventricular tissue, the sinoatrial node, atrioventricular node and Purkinje fibers. Current heart-on-a-chip approaches incorporate biological, electrical, mechanical, and topographical cues to facilitate tissue maturation, therefore improving the predictive power for the chamber-specific therapeutic effects targeting adult human. In this review, we will give a summary of current advances in heart-on-a-chip technology and provide a comprehensive outlook on the challenges involved in the development of human physiologically relevant heart-on-a-chip.

Mapping signalling perturbations in myocardial fibrosis via the integrative phosphoproteomic profiling of tissue from diverse sources.

Study of the molecular basis of myocardial fibrosis is hampered by limited access to tissues from human patients and by confounding variables associated with sample accessibility, collection, processing and storage. Here, we report an integrative strategy based on mass spectrometry for the phosphoproteomic profiling of normal and fibrotic cardiac tissue obtained from surgical explants from patients with hypertrophic cardiomyopathy, from a transaortic-constriction mouse model of cardiac hypertrophy and fibrosis, and from a heart-on-a-chip model of cardiac fibrosis. We used the integrative approach to map the relative abundance of thousands of proteins, phosphoproteins and phosphorylation sites specific to each tissue source, to identify key signalling pathways driving fibrosis and to screen for anti-fibrotic compounds targeting glycogen synthase kinase 3, which has a consistent role as a key mediator of fibrosis in all three types of tissue specimen. The integrative disease-modelling strategy may reveal new insights into mechanisms of cardiac disease and serve as a test bed for drug screening.

A Multimaterial Microphysiological Platform Enabled by Rapid Casting of Elastic Microwires.

Due to escalating drug developmental costs and limitations of cardiotoxicity screening, there is an urgent need to develop robust in vitro 3D tissue culture platforms that can both facilitate the culture of human cardiac tissues and provide noninvasive functional readouts predictive of cardiotoxicity in clinical settings. However, such platforms commonly require complex fabrication procedures that are difficult to scale up to high-throughput testing platforms. Here, innovative multimaterial processing into a scalable and functional platform is proposed in the format of a 96-well plate. Three classes of materials are integrated into the platform. An array of soft elastic microwires is used both as anchors for tissue formation as well as sensors for recording tissue contraction. Conductive carbon electrodes are embedded into the plate to drive electrical stimulation for tissue maturation and pace tissue contraction during drug testing. The bulk of the device is made of rigid polystyrene plastic to eliminate drug-absorbing polydimethylsiloxane (PDMS). The platform has higher throughput than the current state-of-the-art devices, at a significantly reduced cost of manufacturing and tissue production.

Cardiovascular disease models: A game changing paradigm in drug discovery and screening.

Cardiovascular disease is the leading cause of death worldwide. Although investment in drug discovery and development has been sky-rocketing, the number of approved drugs has been declining. Cardiovascular toxicity due to therapeutic drug use claims the highest incidence and severity of adverse drug reactions in late-stage clinical development. Therefore, to address this issue, new, additional, replacement and combinatorial approaches are needed to fill the gap in effective drug discovery and screening. The motivation for developing accurate, predictive models is twofold: first, to study and discover new treatments for cardiac pathologies which are leading in worldwide morbidity and mortality rates; and second, to screen for adverse drug reactions on the heart, a primary risk in drug development. In addition to in vivo animal models, in vitro and in silico models have been recently proposed to mimic the physiological conditions of heart and vasculature. Here, we describe current in vitro, in vivo, and in silico platforms for modelling healthy and pathological cardiac tissues and their advantages and disadvantages for drug screening and discovery applications. We review the pathophysiology and the underlying pathways of different cardiac diseases, as well as the new tools being developed to facilitate their study. We finally suggest a roadmap for employing these non-animal platforms in assessing drug cardiotoxicity and safety.

Biowire Model of Interstitial and Focal Cardiac Fibrosis.

Myocardial fibrosis is a severe global health problem due to its prevalence in all forms of cardiac diseases and direct role in causing heart failure. The discovery of efficient antifibrotic compounds has been hampered due to the lack of a physiologically relevant disease model. Herein, we present a disease model of human myocardial fibrosis and use it to establish a compound screening system. In the Biowire II platform, cardiac tissues are suspended between a pair of poly(octamethylene maleate (anhydride) citrate) (POMaC) wires. Noninvasive functional readouts are realized on the basis of the deflection of the intrinsically fluorescent polymer. The disease model is constructed to recapitulate contractile, biomechanical, and electrophysiological complexities of fibrotic myocardium. Additionally, we constructed a heteropolar integrated model with fibrotic and healthy cardiac tissues coupled together. The integrated model captures the regional heterogeneity of scar lesion, border zone, and adjacent healthy myocardium. Finally, we demonstrate the utility of the system for the evaluation of antifibrotic compounds. The high-fidelity in vitro model system combined with convenient functional readouts could potentially facilitate the development of precision medicine strategies for cardiac fibrosis modeling and establish a pipeline for preclinical compound screening.

High-Content Assessment of Cardiac Function Using Heart-on-a-Chip Devices as Drug Screening Model.

Drug discovery and development continues to be a challenge to the pharmaceutical industry despite great advances in cell and molecular biology that allow for the design of better targeted therapeutics. Many potential drug compounds fail during the clinical trial due to inefficacy and toxicity that were not predicted during preclinical stages. The fundamental problem lies with the use of traditional drug screening models that still largely rely on the use of cell lines or animal cell monolayers, which leads to lack of predictive power of human tissue and organ response to the drug candidates. More physiologically relevant systems are therefore critical in relieving the burden of high failure rates. Emerging knowledge and techniques in tissue engineering and microfabrication have enabled the development of micro-engineered systems - collectively known as organs-on-chips - that may lead to a paradigm shift in preclinical drug screening assays. In this review we explore the technological advances and challenges in the development of heart-on-a-chip models, by addressing current assessment methods for drug-induced cardiotoxicity and providing a perspective on the modifications that should be implemented to realize the full potential of this system.

p53 mediates autophagy and cell death by a mechanism contingent on Bnip3.

Myocardial ischemia and angiotensin II activate the tumor suppressor p53 protein, which promotes cell death. Previously, we showed that the Bcl-2 death gene Bnip3 is highly induced during ischemia, where it triggers mitochondrial perturbations resulting in autophagy and cell death. However, whether p53 regulates Bnip3 and autophagy is unknown. Herein, we provide new compelling evidence for a novel signaling axis that commonly links p53 and Bnip3 for autophagy and cell death. p53 overexpression increased endogenous Bnip3 mRNA and protein levels resulting in mitochondrial defects leading to loss of mitochondrial ΔΨ(m). This was accompanied by an increase in autophagic flux and cell death. Notably, genetic loss of function studies, such as Atg7 knock-down or pharmacological inhibition of autophagy with 3-methyl adenine, suppressed cell death induced by p53--indicating that p53 induces maladaptive autophagy. Our previous work demonstrated that Bnip3 induces mitochondrial defects and autophagic cell death. Conversely, loss of function of Bnip3 in cardiac myocytes or Bnip3(-/-) mouse embryonic fibroblasts prevented mitochondrial targeting of p53, autophagy, and cell death. To our knowledge, these data provide the first evidence for the dual regulation of autophagy and cell death of cardiac myocytes by p53 that is mutually dependent on and obligatorily linked to Bnip3 gene activation. Hence, our findings may explain more fundamentally, how, autophagy and cell death are dually regulated during cardiac stress conditions where p53 is activated.