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The fabrication of complex and stable vasculature in engineered cardiac tissues represents a significant hurdle towards building physiologically relevant models of the heart. Here, we implemented a 3D model of cardiac vasculogenesis, incorporating endothelial cells (EC), stromal cells, and human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CM) in a fibrin hydrogel. The presence of CMs disrupted vessel formation in 3D tissues, resulting in the upregulation of endothelial activation markers and altered extracellular vesicle (EV) signaling in engineered tissues as determined by the proteomic analysis of culture supernatant. miRNA sequencing of CM- and EC-secreted EVs highlighted key EV-miRNAs that were postulated to play differing roles in cardiac vasculogenesis, including the let-7 family and miR-126-3p in EC-EVs. In the absence of CMs, the supplementation of CM-EVs to EC monolayers attenuated EC migration and proliferation and resulted in shorter and more discontinuous self-assembling vessels when applied to 3D vascular tissues. In contrast, supplementation of EC-EVs to the tissue culture media of 3D vascularized cardiac tissues mitigated some of the deleterious effects of CMs on vascular self-assembly, enhancing the average length and continuity of vessel tubes that formed in the presence of CMs. Direct transfection validated the effects of the key EC-EV miRNAs let-7b-5p and miR-126-3p in improving the maintenance of continuous vascular networks. EC-EV supplementation to biofabricated cardiac tissues and microfluidic devices resulted in tissue vascularization, illustrating the use of this approach in the engineering of enhanced, perfusable, microfluidic models of the myocardium., (Creative Commons Attribution license.)

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., Competing Interests: Declaration of interests M.R. and Y.Z. are inventors on an issued patent that describes Biowire technology. This patent is licensed to Valo Health. M.R. and Y.Z. receive licensing revenue., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Ionizable lipid nanoparticles (LNPs) are seeing widespread use in mRNA delivery, notably in SARS-CoV-2 mRNA vaccines. However, the expansion of mRNA therapies beyond COVID-19 is impeded by the absence of LNPs tailored for diverse cell types. In this study, we present the AI-Guided Ionizable Lipid Engineering (AGILE) platform, a synergistic combination of deep learning and combinatorial chemistry. AGILE streamlines ionizable lipid development with efficient library design, in silico lipid screening via deep neural networks, and adaptability to diverse cell lines. Using AGILE, we rapidly design, synthesize, and evaluate ionizable lipids for mRNA delivery, selecting from a vast library. Intriguingly, AGILE reveals cell-specific preferences for ionizable lipids, indicating tailoring for optimal delivery to varying cell types. These highlight AGILE's potential in expediting the development of customized LNPs, addressing the complex needs of mRNA delivery in clinical practice, thereby broadening the scope and efficacy of mRNA therapies., (© 2024. The Author(s).)

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., Competing Interests: Competing interests statement:Y.Z., G.V.-N., B.Z., and M.R. are inventors on patents for cardiac tissue cultivation that are licensed to Valo Health. Q.W., Y.Z., and M.R. have a filed patent application on thermoplastic polymer composition for micro 3D printing and uses thereof. B.Z. holds equity in OrganoBiotech.

Despite tremendous progress in the development of mature heart-on-a-chip models, human cell-based models of myocardial inflammation are lacking. Here, we bioengineered a vascularized heart-on-a-chip with circulating immune cells to model severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-induced acute myocarditis. We observed hallmarks of coronavirus disease (COVID-19)-induced myocardial inflammation, as the presence of immune cells augmented the secretion of proinflammatory cytokines, triggered progressive impairment of contractile function, and altered intracellular calcium transients. An elevation of circulating cell-free mitochondrial DNA (ccf-mtDNA) was measured first in the heart-on-a-chip and then validated in COVID-19 patients with low left ventricular ejection fraction, demonstrating that mitochondrial damage is an important pathophysiological hallmark of inflammation-induced cardiac dysfunction. Leveraging this platform in the context of SARS-CoV-2-induced myocardial inflammation, we established that administration of endothelial cell-derived exosomes effectively rescued the contractile deficit, normalized calcium handling, elevated the contraction force, and reduced the ccf-mtDNA and cytokine release via Toll-like receptor-nuclear factor κB signaling axis.

Ionizable lipid nanoparticles (LNPs) pivotal to the success of COVID-19 mRNA (messenger RNA) vaccines hold substantial promise for expanding the landscape of mRNA-based therapies. Nevertheless, the risk of mRNA delivery to off-target tissues highlights the necessity for LNPs with enhanced tissue selectivity. The intricate nature of biological systems and inadequate knowledge of lipid structure-activity relationships emphasize the significance of high-throughput methods to produce chemically diverse lipid libraries for mRNA delivery screening. Here, we introduce a streamlined approach for the rapid design and synthesis of combinatorial libraries of biodegradable ionizable lipids. This led to the identification of iso-A11B5C1, an ionizable lipid uniquely apt for muscle-specific mRNA delivery. It manifested high transfection efficiencies in muscle tissues, while significantly diminishing off-targeting in organs like the liver and spleen. Moreover, iso-A11B5C1 also exhibited reduced mRNA transfection potency in lymph nodes and antigen-presenting cells, prompting investigation into the influence of direct immune cell transfection via LNPs on mRNA vaccine effectiveness. In comparison with SM-102, while iso-A11B5C1's limited immune transfection attenuated its ability to elicit humoral immunity, it remained highly effective in triggering cellular immune responses after intramuscular administration, which is further corroborated by its strong therapeutic performance as cancer vaccine in a melanoma model. Collectively, our study not only enriches the high-throughput toolkit for generating tissue-specific ionizable lipids but also encourages a reassessment of prevailing paradigms in mRNA vaccine design. This study encourages rethinking of mRNA vaccine design principles, suggesting that achieving high immune cell transfection might not be the sole criterion for developing effective mRNA vaccines., Competing Interests: Competing interests statement:J.C., Y.X., and B.L. have filed a provisional patent for the development of the described lipids.

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., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Peter H. Backx reports financial support was provided by Canadian Institutes for Health Research. Peter H. Backx reports financial support was provided by Peter Munk Cardiac Centre Innovation Committee Award. Kumaraswamy Nanthakumar reports financial support was provided by Heart and Stroke Foundation of Ontario. Brian J. Cox reports financial support was provided by Canadian Institutes for Health Research. Milica Radisic reports financial support was provided by Canadian Institutes for Health Research. Milica Radisic reports financial support was provided by National Institutes of Health. Yimu Zhao reports financial support was provided by Canadian Institutes for Health Research. Milica Radisic reports financial support and equipment, drugs, or supplies were provided by Canada Foundation for Innovation and the Ontario Research Fund. Milica Radisic is an inventor on a patent covering biowire heart-on-a-chip technology which is licensed to Valo Health. She receives royalties from Valo Health. Yimu Zhao is an inventor on a patent covering biowire heart-on-a-chip technology which is licensed to Valo Health. She receives royalties from Valo Health., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Coronavirus disease 2019 (COVID-19) has been a major global health concern since its emergence in 2019, with over 680 million confirmed cases as of April 2023. While COVID-19 has been strongly associated with the development of cardiovascular complications, the specific mechanisms by which viral infection induces myocardial dysfunction remain largely controversial as studies have shown that the severe acute respiratory syndrome coronavirus-2 can lead to heart failure both directly, by causing damage to the heart cells, and indirectly, by triggering an inflammatory response throughout the body. In this review, we summarize the current understanding of potential mechanisms that drive heart failure based on in vitro studies. We also discuss the significance of three-dimensional heart-on-a-chip technology in the context of the current and future pandemics., Competing Interests: M.R. and Y.Z. are inventors on a patent describing Biowire II heart‐on‐a‐chip technology that is licensed to Valo Health. They receive royalty payments. The remaining author declares no conflicts of interest., (© 2023 The Authors. Bioengineering & Translational Medicine published by Wiley Periodicals LLC on behalf of American Institute of Chemical Engineers.)

Coronavirus disease 2019 (COVID-19) was primarily identified as a novel disease causing acute respiratory syndrome. However, as the pandemic progressed various cases of secondary organ infection and damage by severe respiratory syndrome coronavirus 2 (SARS-CoV-2) have been reported, including a breakdown of the vascular barrier. As SARS-CoV-2 gains access to blood circulation through the lungs, the virus is first encountered by the layer of endothelial cells and immune cells that participate in host defense. Here, we developed an approach to study SARS-CoV-2 infection using vasculature-on-a-chip. We first modeled the interaction of virus alone with the endothelialized vasculature-on-a-chip, followed by the studies of the interaction of the virus exposed-endothelial cells with peripheral blood mononuclear cells (PBMCs). In an endothelial model grown on a permeable microfluidic bioscaffold under flow conditions, both human coronavirus (HCoV)-NL63 and SARS-CoV-2 presence diminished endothelial barrier function by disrupting VE-cadherin junctions and elevating the level of pro-inflammatory cytokines such as interleukin (IL)-6, IL-8, and angiopoietin-2. Inflammatory cytokine markers were markedly more elevated upon SARS-CoV-2 infection compared to HCoV-NL63 infection. Introduction of PBMCs with monocytes into the vasculature-on-a-chip upon SARS-CoV-2 infection further exacerbated cytokine-induced endothelial dysfunction, demonstrating the compounding effects of inter-cellular crosstalk between endothelial cells and monocytes in facilitating the hyperinflammatory state. Considering the harmful effects of SARS-CoV-2 on endothelial cells, even without active virus proliferation inside the cells, a potential therapeutic approach is critical. We identified angiopoietin-1 derived peptide, QHREDGS, as a potential therapeutic capable of profoundly attenuating the inflammatory state of the cells consistent with the levels in non-infected controls, thereby improving the barrier function and endothelial cell survival against SARS-CoV-2 infection in the presence of PBMC.

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., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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.

Despite showing a great promise in the field of nanomedicine, nanoparticles have gained a significant attention from regulatory agencies regarding their possible adverse health effects upon environmental exposure. Whether those nanoparticles are generated through intentional or unintentional means, the constant exposure to nanomaterials can inevitably lead to unintended consequences based on epidemiological data, yet the current understanding of nanotoxicity is insufficient relative to the rate of their emission in the environment and the lack of predictive platforms that mimic the human physiology. This calls for a development of more physiologically relevant models, which permit the comprehensive and systematic examination of toxic properties of nanoparticles. With the advancement in microfabrication techniques, scientists have shifted their focus on the development of an engineered system that acts as an intermediate between a well-plate system and animal models, known as organ-on-a-chips. The ability of organ-on-a-chip models to recapitulate in vivo like microenvironment and responses offers a new avenue for nanotoxicological research. In this review, we aim to provide overview of assessing potential risks of nanoparticle exposure using organ-on-a-chip systems and their potential to delineate biological mechanisms of epidemiological findings., Competing Interests: M.R. is a co-founder of TARA Biosystems and holds euiqty in this company., (© 2021 The Authors. Production and hosting by Elsevier B.V. on behalf of KeAi Communications Co., Ltd.)

Kidney-on-a-chip devices may revolutionize the discovery of new therapies. However, fabricating a 3D glomerulus remains a challenge, due to a requirement for a microscale soft material with complex topography to support cell culture in a native configuration. Here, we describe the use of microfluidic spinning to recapitulate complex concave and convex topographies over multiple length scales, required for biofabrication of a biomimetic 3D glomerulus. We produced a microfluidic extruded topographic hollow fiber (h-FIBER), consisting of a vessel-like perfusable tubular channel for endothelial cell cultivation, and a glomerulus-like knot with microconvex topography on its surface for podocyte cultivation. Meter long h-FIBERs were produced in microfluidics within minutes, followed by chemically induced inflation for generation of topographical cues on the 3D scaffold surface. The h-FIBERs were assembled into a hot-embossed plastic 96-well plate. Long-term perfusion, podocyte barrier formation, endothelialization, and permeability tests were easily performed by a standard pipetting technique on the platform. Following long-term culture (1 month), a functional filtration barrier, measured by the transfer of albumin from the blood vessel side to the ultrafiltrate side, suggested the establishment of an engineered glomerulus., Competing Interests: The authors declare no competing financial interest., (Copyright © 2020 American Chemical Society.)

Objective: Apply high-resolution benchtop micro-computed tomography (micro-CT) to gain greater understanding and knowledge of the formation of the micro-damage process zone formed during traverse fracture of cortical bone., Methods: Bovine cortical bone was cut into single edge notch (bending) fracture testing specimens with the crack on the transverse plane and oriented to grow in the circumferential direction. We used a multi-specimen technique and deformed the specimens to various individual secant modulus loss levels (P-values) up to and including maximum load (Pmax). Next, the specimens were infiltrated with a BaSO 4 precipitation stain and scanned at 3.57-μm isotropic voxel size using a benchtop high resolution-micro-CT. Measurements of the micro-damage process zone volume, width and height were made. These were compared with the simple Irwin's process zone model and with finite element models. Electron and confocal microscopy confirmed the formation of BaSO 4 precipitate in micro-cracks and other porosity, and an interesting novel mechanism similar to tunneling., Results: Measurable micro-damage was detected at low P values and the volume of the process zone increased according to a second order polynomial trend. Both width and height grew linearly up to Pmax, at which point the process zone cross-section (perpendicular to the plane of the crack) was almost circular on average with a radius of approximately 550µm (approximately one quarter of the unbroken ligament thickness) and corresponding to the shape expected for a biological composite under plane stress conditions., Conclusion: This study reports details of the micro-damage fracture process zone previously unreported for cortical bone. High-resolution micro-CT enables 3D visualization and measurement of the process zone and confirmation that the crack front edge and process zone are affected by microstructure. It is clear that the process zone for the specimens studied grows to be meaningfully large, confirming the need for the J-integral approach and it does not achieve steady state at Pmax in most specimens. With further development, this approach may become valuable towards better understanding the role of the process zone in cortical bone fracture and the effects of relevant modifications towards changes in fracture toughness in a cost effective way., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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.