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Microfabricated perfusable cardiac biowire: A platform that mimics native cardiac bundle
Abstract and Figures
Tissue engineering enables the generation of three-dimensional (3D) functional cardiac tissue for pre-clinical testing in vitro, which is critical for new drug development. However, current tissue engineering methods poorly recapitulate the architecture of oriented cardiac bundles with supporting capillaries. In this study, we designed a microfabricated bioreactor to generate 3D micro-tissues, termed biowires, using both primary neonatal rat cardiomyocytes and human embryonic stem cell (hESC) derived cardiomyocytes. Perfusable cardiac biowires were generated with polytetrafluoroethylene (PTFE) tubing template, and were integrated with electrical field stimulation using carbon rod electrodes. To demonstrate the feasibility of this platform for pharmaceutical testing, nitric oxide (NO) was released from perfused sodium nitroprusside (SNP) solution and diffused through the tubing. The NO treatment slowed down the spontaneous beating of cardiac biowires based on hESC derived cardiomyocytes and degraded the myofibrillar cytoskeleton of the cardiomyocytes within the biowires. The biowires were also integrated with electrical stimulation using carbon rod electrodes to further improve phenotype of cardiomyocytes, as indicated by organized contractile apparatus, higher Young's modulus, and improved electrical properties. This microfabricated platform provides a unique opportunity to assess pharmacological effects on cardiac tissue in vitro by perfusion in a cardiac bundle model, which could provide improved physiological relevance.
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... Besides 3D cardiac MPS, other common 3D cardiac in vitro platforms include engineered cardiac tissues consisting of micropillars, micro posts, or cantilevers to evaluate contractility (e.g. biowire, mantarray, myrTissue) (Dou et al., 2022;Veldhuizen et al., 2019;Xiao et al., 2014). Cardiac MPS open the possibility to study cardiac physiology, drug responses, and disease mechanisms in a controlled setting, which offers advantages over traditional cell culture and animal models. ...
Microphysiological systems (MPS) are complex in vitro tools that incorporate cells derived from various healthy or disease-state human or animal tissues and organs. While MPS have limitations, including a lack of globally harmonized guidelines for standardization, they have already proven impactful in certain areas of drug development. Further research and regulatory acceptance of MPS will contribute to making them even more effective tools in the future. This review explores the potential applications of human liver, gut, lung, and cardiac MPS in drug development, focusing on disease modeling, safety assessment, and pharmacokinetic studies. Various technical parameters and relevant endpoints for system assessment are discussed alongside challenges such as cell sourcing, reproducibility, and the integration of multiple tissues or organs. The importance of collaborative efforts between academia, industry, and regulatory agencies to develop standardized protocols and validation criteria is emphasized. With ongoing advancements and cooperative initiatives, MPS are poised to play a significant role in enhancing the predictivity and reliability of nonclinical testing, thereby transforming drug development and regulatory processes.
... The ideal characteristics of a biomaterial include being biocompatible to support cell attachment, porous to support gas and nutrient exchange, and eventually degradable. Common biomaterials used to facilitate tissue engineering include natural components of the ECM such as collagen, [19][20][21][22] fibrin, 23 gelatin, 24 chitosan, 25 alginate, 26 and synthetic biomaterials. 27,28 While naturally occurring biomaterials often promote cell attachment and can degrade in a nontoxic manner, synthetic biomaterials are more amenable to chemical modifications to improve degradation rates, precisely tailor mechanical properties, and provide consistency between batches. ...
Cardiovascular diseases are the leading cause of morbidity and mortality worldwide with numerous inflammatory cell etiologies associated with impaired cardiac function and heart failure. Inflammatory cardiomyopathy, also known as myocarditis, is an acquired cardiomyopathy characterized by inflammatory cell infiltration into the myocardium with a high risk of progression to deteriorated cardiac function. Recently, amidst the ongoing COVID-19 pandemic, the emergence of acute myocarditis as a complication of SARS-CoV-2 has garnered significant concern. Given its mechanisms remain elusive in conjunction with the recent withdrawal of previously FDA-approved antiviral therapeutics and prophylactics due to unexpected cardiotoxicity, there is a pressing need for human-mimetic platforms to investigate disease pathogenesis, model dysfunctional features, and support pre-clinical drug screening. Traditional in vitro models for studying cardiovascular diseases have inherent limitations in recapitulating the complexity of the in vivo microenvironment. Heart-on-a-chip technologies, combining microfabrication, microfluidics, and tissue engineering techniques, have emerged as a promising approach for modeling inflammatory cardiac diseases like myocarditis. This review outlines the established and emerging conditions of inflamed myocardium, identifying key features essential for recapitulating inflamed myocardial structure and functions in heart-on-a-chip models, highlighting recent advancements, including the integration of anisotropic contractile geometry, cardiomyocyte maturity, electromechanical functions, vascularization, circulating immunity, and patient/sex specificity. Finally, we discuss the limitations and future perspectives necessary for the clinical translation of these advanced technologies.
... Multi-organ-on-a-chip is a biomimetic platform that utilizes microfluidic chip and three-dimension (3D) cell culture to model human physiology and pathology in vitro. Since the publication of a lung-on-a-chip in 2010 [1], this field has seen over a decade of development, resulting in various organ-specific chips, including liver-on-a-chip [2][3][4], intestine-on-a-chip [2,[5][6][7], brain-on-a-chip [8][9][10], heart-on-a-chip [11,12], etc. These chips have already shown great potential for drug discovery and precision medicine. ...
Multi-organ chips are effective at emulating human tissue and organ functions and at replicating the interactions among tissues and organs. An arrayed brain–heart chip was introduced whose configuration comprises open culture chambers and closed biomimetic vascular channels distributed in a horizontal pattern, separated from each other by an endothelial barrier based on fibrin matrix. A 300 μm-high and 13.2 mm-long endothelial barrier surrounded each organoid culture chamber, thereby satisfying the material transport requirements. Numerical simulations were used to analyze the construction process of fibrin barriers in order to optimize the structural design and experimental manipulation, which exhibited a high degree of correlation with experiment results. In each interconnective unit, a cerebral organoid, a cardiac organoid, and endothelial cells were co-cultured stably for a minimum of one week. The permeability of the endothelial barrier and recirculating perfusion enabled cross talk between cerebral organoids and cardiac organoids, as well as between organoids and endothelial cells. This was corroborated by the presence of cardiac troponin I (cTnI) in the cerebral organoid culture chamber and the observation of cerebral organoid and endothelial cells invading the fibrin matrix after one week of co-culture. The arrayed chip was simple to manipulate, clearly visible under a microscope, and compatible with automated pipetting devices, and therefore had significant potential for application.
Organ-on-a-chip (OoC) devices represent advanced in vitro models that enable mimicking the human tissue architecture function and physiology, offering a promising alternative to traditional animal testing methods. These devices combine the microfluidics with soft materials, specifically hydrogel membranes (HMs) for mimicking the extracellular matrix (ECM) and biological barriers, such as the blood-brain barrier (BBB). Hydrogels are ideal biomaterials for OoC systems because of their tunable properties, comprising biocompatibility, biodegradability, and microscale self-assembly. The integration of HMs with OoC devices provides an effective way to create dynamic, biologically relevant environments that supporting living cells for applications such as drug discovery, disease modeling, and personalized medicine. Recent advancements in fabrication technologies such as 3D printing, photolithography, and bioprinting have additionally advanced development of such systems. This review surveys the role of HMs in OoC platforms, highlighting their material properties, self-assembly behavior, and challenges in fabrication. Additionally, we discuss the progress made in the past five years in utilizing HMs for applications in tissue engineering, drug development, and biosensing, with a focus on their interface dynamics and structural self-organization. The future perspective on OoC technology has also been patterned to provide a broader image on integration of OoC with personalized medicine and advanced drug delivery systems.
Cigarette smoking is positively and robustly associated with cardiovascular disease (CVD), including hypertension, atherosclerosis, cardiac arrhythmias, stroke, thromboembolism, myocardial infarctions, and heart failure. However, after more than a decade of ENDS presence in the U.S. marketplace, uncertainty persists regarding the long-term health consequences of ENDS use for CVD. New approach methods (NAMs) in the field of toxicology are being developed to enhance rapid prediction of human health hazards. Recent technical advances can now consider impact of biological factors such as sex and race/ethnicity, permitting application of NAMs findings to health equity and environmental justice issues. This has been the case for hazard assessments of drugs and environmental chemicals in areas such as cardiovascular, respiratory, and developmental toxicity. Despite these advances, a shortage of widely accepted methodologies to predict the impact of ENDS use on human health slows the application of regulatory oversight and the protection of public health. Minimizing the time between the emergence of risk (e.g., ENDS use) and the administration of well-founded regulatory policy requires thoughtful consideration of the currently available sources of data, their applicability to the prediction of health outcomes, and whether these available data streams are enough to support an actionable decision. This challenge forms the basis of this white paper on how best to reveal potential toxicities of ENDS use in the human cardiovascular system—a primary target of conventional tobacco smoking. We identify current approaches used to evaluate the impacts of tobacco on cardiovascular health, in particular emerging techniques that replace, reduce, and refine slower and more costly animal models with NAMs platforms that can be applied to tobacco regulatory science. The limitations of these emerging platforms are addressed, and systems biology approaches to close the knowledge gap between traditional models and NAMs are proposed. It is hoped that these suggestions and their adoption within the greater scientific community will result in fresh data streams that will support and enhance the scientific evaluation and subsequent decision-making of tobacco regulatory agencies worldwide.
Graphical Abstract
Models for Cardiovascular Toxicity Testing E-cigarettes and nicotine delivery systems can be examined using multiple model systems. In silico models might predict adverse cardiovascular effects that can be screened for using 2-D and 3-D models using induced pluripotent stem cell (iPSC) derived cardiac tissue and “omics” profiling such as single-cell RNA sequencing (scRNA-seq) or high-throughput functional analysis with a multiple electrode array (MEA). Biologic plausibility of detected effects can be corroborated using ex vivo or in vivo models, which may also lead to the discovery of new biomarkers or treatments for CVD.
Modeling human pregnancy is challenging as two subjects, the mother and fetus, must be evaluated in tandem. To understand pregnancy, parturition, and adverse pregnancy outcomes, the two feto-maternal interfaces (FMi) that form during gestation (i.e., the placenta and fetal membrane) need to be investigated to understand their biological roles, and organ dysfunction can lead to adverse outcomes. Adverse pregnancy outcomes such as preterm rupture of the membranes, spontaneous preterm birth, preeclampsia, intra-uterine growth restriction, and gestational diabetes rates are on the rise worldwide, highlighting the need for future studies and a better understanding of molecular and cellular pathways that contribute to disease onset. Current in vivo animal models nor in vitro cell culture systems can answer these questions as they do not model the function or structure of human FMis. Utilizing microfabrication and soft-lithography techniques, microfluidic organ-on-chip (OOC) devices have been adapted by many fields to model the anatomy and biological function of complex organs and organ systems within small in vitro platforms.
These techniques have been adapted to recreate the fetal membrane FMi (FMi-OOC) using immortalized cells and collagen derived from patient samples. The FMi-OOC is a four-cell culture chamber, concentric circle system, that contains both fetal (amniochorion) and maternal (decidua) cellular layers and has been validated to model physiological and pathological states of pregnancy (i.e., ascending infection, systemic oxidative stress, and maternal toxicant exposure). This platform is fully compatible with various analytical methods such as microscopy and biochemical analysis. This protocol will outline this device’s fabrication, cell loading, and utility to model ascending infection-related adverse pregnancy outcomes.
Directed differentiation protocols enable derivation of cardiomyocytes from human pluripotent stem cells (hPSCs) and permit engineering of human myocardium in vitro. However, hPSC-derived cardiomyocytes are reflective of very early human development, limiting their utility in the generation of in vitro models of mature myocardium. Here we describe a platform that combines three-dimensional cell cultivation with electrical stimulation to mature hPSC-derived cardiac tissues. We used quantitative structural, molecular and electrophysiological analyses to explain the responses of immature human myocardium to electrical stimulation and pacing. We demonstrated that the engineered platform allows for the generation of three-dimensional, aligned cardiac tissues (biowires) with frequent striations. Biowires submitted to electrical stimulation had markedly increased myofibril ultrastructural organization, elevated conduction velocity and improved both electrophysiological and Ca(2+) handling properties compared to nonstimulated controls. These changes were in agreement with cardiomyocyte maturation and were dependent on the stimulation rate.
The mammalian heart has little capacity to regenerate, and following injury the myocardium is replaced by non-contractile scar tissue. Consequently, increased wall stress and workload on the remaining myocardium leads to chamber dilation, dysfunction, and heart failure. Cell-based therapy with an autologous, epigenetically reprogrammed, and cardiac-committed progenitor cell source could potentially reverse this process by replacing the damaged myocardium with functional tissue. However, it is unclear whether cardiac progenitor cell-derived cardiomyocytes are capable of attaining levels of structural and functional maturity comparable to that of terminally-fated cardiomyocytes. Here, we first describe the derivation of mouse induced pluripotent stem (iPS) cells, which once differentiated allow for the enrichment of Nkx2-5(+) cardiac progenitors, and the cardiomyocyte-specific expression of the red fluorescent protein. We show that the cardiac progenitors are multipotent and capable of differentiating into endothelial cells, smooth muscle cells and cardiomyocytes. Moreover, cardiac progenitor selection corresponds to cKit(+) cell enrichment, while cardiomyocyte cell-lineage commitment is concomitant with dual expression of either cKit/Flk1 or cKit/Sca-1. We proceed to show that the cardiac progenitor-derived cardiomyocytes are capable of forming electrically and mechanically coupled large-scale 2D cell cultures with mature electrophysiological properties. Finally, we examine the cell progenitors' ability to form electromechanically coherent macroscopic tissues, using a physiologically relevant 3D culture model and demonstrate that following long-term culture the cardiomyocytes align, and form robust electromechanical connections throughout the volume of the biosynthetic tissue construct. We conclude that the iPS cell-derived cardiac progenitors are a robust cell source for tissue engineering applications and a 3D culture platform for pharmacological screening and drug development studies.
Oxidative damage from elevated production of reactive oxygen species (ROS) contributes to ischemia-reperfusion injury in myocardial infarction and stroke. The mechanism by which the increase in ROS occurs is not known, and it is unclear how this increase can be prevented. A wide variety of nitric oxide donors and S-nitrosating agents protect the ischemic myocardium from infarction, but the responsible mechanisms are unclear. Here we used a mitochondria-selective S-nitrosating agent, MitoSNO, to determine how mitochondrial S-nitrosation at the reperfusion phase of myocardial infarction is cardioprotective in vivo in mice. We found that protection is due to the S-nitrosation of mitochondrial complex I, which is the entry point for electrons from NADH into the respiratory chain. Reversible S-nitrosation of complex I slows the reactivation of mitochondria during the crucial first minutes of the reperfusion of ischemic tissue, thereby decreasing ROS production, oxidative damage and tissue necrosis. Inhibition of complex I is afforded by the selective S-nitrosation of Cys39 on the ND3 subunit, which becomes susceptible to modification only after ischemia. Our results identify rapid complex I reactivation as a central pathological feature of ischemia-reperfusion injury and show that preventing this reactivation by modification of a cysteine switch is a robust cardioprotective mechanism and hence a rational therapeutic strategy.
Artificial reconstruction of fibre-shaped cellular constructs could greatly contribute to tissue assembly in vitro. Here we show that, by using a microfluidic device with double-coaxial laminar flow, metre-long core-shell hydrogel microfibres encapsulating ECM proteins and differentiated cells or somatic stem cells can be fabricated, and that the microfibres reconstitute intrinsic morphologies and functions of living tissues. We also show that these functional fibres can be assembled, by weaving and reeling, into macroscopic cellular structures with various spatial patterns. Moreover, fibres encapsulating primary pancreatic islet cells and transplanted through a microcatheter into the subrenal capsular space of diabetic mice normalized blood glucose concentrations for about two weeks. These microfibres may find use as templates for the reconstruction of fibre-shaped functional tissues that mimic muscle fibres, blood vessels or nerve networks in vivo.
Recent progress in cell transplantation therapy to repair impaired hearts has encouraged further attempts to bioengineer 3-dimensional (3-D) heart tissue from cultured cardiomyocytes. Cardiac tissue engineering is currently pursued utilizing conventional technology to fabricate 3-D biodegradable scaffolds as a temporary extracellular matrix. By contrast, new methods are now described to fabricate pulsatile cardiac grafts using new technology that layers cell sheets 3-dimensionally. We apply novel cell culture surfaces grafted with temperature-responsive polymer, poly(N-isopropylacrylamide) (PIPAAm), from which confluent cells detach as a cell sheet simply by reducing temperature without any enzymatic treatments. Neonatal rat cardiomyocyte sheets detached from PIPAAm-grafted surfaces were overlaid to construct cardiac grafts. Layered cell sheets began to pulse simultaneously and morphological communication via connexin43 was established between the sheets. When 4 sheets were layered, engineered constructs were macroscopically observed to pulse spontaneously. In vivo, layered cardiomyocyte sheets were transplanted into subcutaneous tissues of nude rats. Three weeks after transplantation, surface electrograms originating from transplanted grafts were detected and spontaneous beating was macroscopically observed. Histological studies showed characteristic structures of heart tissue and multiple neovascularization within contractile tissues. Constructs transplanted into 3-week-old rats exhibited more cardiomyocyte hypertrophy and less connective tissue than those placed into 8-week-old rats. Long-term survival of pulsatile cardiac grafts was confirmed up to 12 weeks. These results demonstrate that electrically communicative pulsatile 3-D cardiac constructs were achieved both in vitro and in vivo by layering cardiomyocyte sheets. Cardiac tissue engineering based on this technology may prove useful for heart model fabrication and cardiovascular tissue repair. The full text of this article is available at http://www.circresaha.org.
We present the design of a higher throughput "heart on a chip" which utilizes a semi-automated fabrication technique to process sub millimeter sized thin film cantilevers of soft elastomers. Anisotropic cardiac microtissues which recapitulate the laminar architecture of the heart ventricle are engineered on these cantilevers. Deflection of these cantilevers, termed Muscular Thin Films (MTFs), during muscle contraction allows calculation of diastolic and systolic stresses generated by the engineered tissues. We also present the design of a reusable one channel fluidic microdevice completely built out of autoclavable materials which incorporates various features required for an optical cardiac contractility assay: metallic base which fits on a heating element for temperature control, transparent top for recording cantilever deformation and embedded electrodes for electrical field stimulation of the tissue. We employ the microdevice to test the positive inotropic effect of isoproterenol on cardiac contractility at dosages ranging from 1 nM to 100 μM. The higher throughput fluidic heart on a chip has applications in testing of cardiac tissues built from rare or expensive cell sources and for integration with other organ mimics. These advances will help alleviate translational barriers for commercial adoption of these technologies by improving the throughput and reproducibility of readout, standardization of the platform and scalability of manufacture.
Induced pluripotent stem cells offer the possibility to generate patient-specific stem cell lines from individuals affected by inherited disorders. Cardiomyocytes differentiated from such patient-specific induced pluripotent stem cells lines have been used to study the pathophysiology of arrhythmogenic heart diseases, such as the long-QT syndrome or catecholaminergic polymorphic ventricular tachycardia. Testing for unwanted drug side effects or tailoring medical treatment to the specific needs of individual patients with arrhythmogenic disorders may become future applications of this emerging technology.
Each year, the American Heart Association (AHA), in conjunction with the Centers for Disease Control and Prevention, the National Institutes of Health, and other government agencies, brings together the most up-to-date statistics on heart disease, stroke, other vascular diseases, and their risk factors and presents them in its Heart Disease and Stroke Statistical Update. The Statistical Update is a critical resource for researchers, clinicians, healthcare policy makers, media professionals, the lay public, and many others who seek the best available national data on heart disease, stroke, and other cardiovascular disease-related morbidity and mortality and the risks, quality of care, use of medical procedures and operations, and costs associated with the management of these diseases in a single document. Indeed, since 1999, the Statistical Update has been cited >10 500 times in the literature, based on citations of all annual versions. In 2012 alone, the various Statistical Updates were cited ≈3500 times (data from Google Scholar). In recent years, the Statistical Update has undergone some major changes with the addition of new chapters and major updates across multiple areas, as well as increasing the number of ways to access and use the information assembled. For this year's edition, the Statistics Committee, which produces the document for the AHA, updated all of the current chapters with the most recent nationally representative data and inclusion of relevant articles from the literature over the past year. This year's edition includes a new chapter on peripheral artery disease, as well as new data on the monitoring and benefits of cardiovascular health in the population, with additional new focus on evidence-based approaches to changing behaviors, implementation strategies, and implications of the AHA's 2020 Impact Goals. Below are a few highlights from this year's Update.