Fig 1 - uploaded by Yun Xiao
Content may be subject to copyright.
Cardiac bundles in native myocardium. (a) Schematic illustration of the structure of cardiac bundles in native myocardium. Cardiomyocytes are elongated, aligned, and grouped into bundles around capillaries. (b) Tangential section of adult rat myocardium with CD31 staining (brown). Nuclei were counterstained as light violet (long arrows). The blood vessels were noted as asterisks while the capillaries were noted as short arrows. (c) Fluorescent image of tangential cryosection of neonatal rat myocardium. Cardiac troponin T (cTnT) was stained against Alexa 488-labeled (green) antibody, showing its unique striation structure. Cell nuclei were counterstained with DAPI (blue) and the long arrows indicate elongated nuclei.
Source publication
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 microfabri...
Contexts in source publication
Context 1
... validate our microfabricated bioreactor, neonatal rat cardiomyocytes were used in preliminary studies. Only when seeded at higher cell density (>5 × 10 7 cells ml −1 ), which is comparable to the cell density in the native rat myocardium (~10 8 cells ml −1 ), 37 the cardiac biowires started spontaneous beating on day 3-4. The template provided contact guidance for the cells to elongate and align along with, recapitulating the anisotropic properties of cardiomyocytes in the native myocardium. The image analysis was done on cell nuclei due to the difficulty of defining cell membranes within 3D tissue. However, nuclear alignment is a sufficient indication of cell alignment and also one of the hallmarks of native myocar- dium (Fig. ...
Context 2
... native myocardium consists of spatially well-defined cardiac bundles with supporting vasculature (Fig. 1a) and the cardiomyocytes within the cardiac bundles are highly anisotropic (Fig. 1b). In this study, we have developed a microfabricated bioreactor to generate cardiac biowires in vitro recapitulating the structure and function of native cardiac bundles. To the best of our knowledge, this is the first study to examine the drug effects on cardiomyocytes by perfusion in a cardiac bundle model, which better mimics native myocardium mass transfer properties compared to other engineered heart tissues. This bioreactor provided topo- graphical cues for the cardiac cells to elongate and align, and was also integrated with other cues, e.g. electrical ...
Context 3
... native myocardium consists of spatially well-defined cardiac bundles with supporting vasculature (Fig. 1a) and the cardiomyocytes within the cardiac bundles are highly anisotropic (Fig. 1b). In this study, we have developed a microfabricated bioreactor to generate cardiac biowires in vitro recapitulating the structure and function of native cardiac bundles. To the best of our knowledge, this is the first study to examine the drug effects on cardiomyocytes by perfusion in a cardiac bundle model, which better mimics native myocardium mass transfer properties compared to other engineered heart tissues. This bioreactor provided topo- graphical cues for the cardiac cells to elongate and align, and was also integrated with other cues, e.g. electrical ...
Context 4
... native myocardium has a highly anisotropic structure (Fig. 1a) with a high density of capillaries (Fig. 1b) and surrounding elongated and aligned cells (Fig. 1c). In the native heart, extracellular matrix (ECM) serves as a template for cells to align and elongate. 28 In addition, a structural correlation between directionality of capillaries and cardiomyocytes can be readily observed. 6 We aimed to emulate this in our biowire bio- reactor by introducing a perfusable tubing template and by using a hydrogel, for cell seeding, consisting of ECM molecules normally present in the native heart. Primary neonatal rat cardiomyocytes were used to generate 3D, self-assembled car- diac biowires by seeding within type I collagen-based gel into microfabricated PDMS platforms with suspended templates (Fig. 2b). Seeded cells remodeled and contracted the collagen gel matrix around the templates within a week (Fig. 2a, 4a). The gel compaction only occurred with the presence of the seeded cells, as cell-free gels did not compact or degrade during the culture time, and the compaction rate positively correlated with the cell seeding density (Fig. 2d). Cardiac biowires of different dimensions could be generated by customizing the dimensions of the biowire bioreactor. Here, we generated biowires as long as 5 cm (Fig. 2c). Generation of longer biowires might be possible; however it was not explored in this ...
Context 5
... native myocardium has a highly anisotropic structure (Fig. 1a) with a high density of capillaries (Fig. 1b) and surrounding elongated and aligned cells (Fig. 1c). In the native heart, extracellular matrix (ECM) serves as a template for cells to align and elongate. 28 In addition, a structural correlation between directionality of capillaries and cardiomyocytes can be readily observed. 6 We aimed to emulate this in our biowire bio- reactor by introducing a perfusable tubing template and by using a hydrogel, for cell seeding, consisting of ECM molecules normally present in the native heart. Primary neonatal rat cardiomyocytes were used to generate 3D, self-assembled car- diac biowires by seeding within type I collagen-based gel into microfabricated PDMS platforms with suspended templates (Fig. 2b). Seeded cells remodeled and contracted the collagen gel matrix around the templates within a week (Fig. 2a, 4a). The gel compaction only occurred with the presence of the seeded cells, as cell-free gels did not compact or degrade during the culture time, and the compaction rate positively correlated with the cell seeding density (Fig. 2d). Cardiac biowires of different dimensions could be generated by customizing the dimensions of the biowire bioreactor. Here, we generated biowires as long as 5 cm (Fig. 2c). Generation of longer biowires might be possible; however it was not explored in this ...
Context 6
... native myocardium has a highly anisotropic structure (Fig. 1a) with a high density of capillaries (Fig. 1b) and surrounding elongated and aligned cells (Fig. 1c). In the native heart, extracellular matrix (ECM) serves as a template for cells to align and elongate. 28 In addition, a structural correlation between directionality of capillaries and cardiomyocytes can be readily observed. 6 We aimed to emulate this in our biowire bio- reactor by introducing a perfusable tubing template and by using a hydrogel, for cell seeding, consisting of ECM molecules normally present in the native heart. Primary neonatal rat cardiomyocytes were used to generate 3D, self-assembled car- diac biowires by seeding within type I collagen-based gel into microfabricated PDMS platforms with suspended templates (Fig. 2b). Seeded cells remodeled and contracted the collagen gel matrix around the templates within a week (Fig. 2a, 4a). The gel compaction only occurred with the presence of the seeded cells, as cell-free gels did not compact or degrade during the culture time, and the compaction rate positively correlated with the cell seeding density (Fig. 2d). Cardiac biowires of different dimensions could be generated by customizing the dimensions of the biowire bioreactor. Here, we generated biowires as long as 5 cm (Fig. 2c). Generation of longer biowires might be possible; however it was not explored in this ...
Citations
... 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.
... In general, hPSC-CMs are mixed with fibroblasts (and/or other cardiac cells) in hydrogels and then poured into casting molds [27,28]. Since the development of the first EHTs two decades ago, a series of tissue models have been produced with diverse geometries and formats such as cardiac biowires [29,30], microwires [31], patches [32], tubeshaped EHTs [33], ring-shaped EHTs [34,35], and chamberforming ventricle models [36,37]. Many of these EHT models can incorporate genetically encoded calcium and voltage indicators with laser-confocal microscopy and optical mapping technology to enable simultaneous analyses of contractile force, electrophysiological properties, and calcium dynamics [27]. ...
Cardiovascular research has heavily relied on studies using patient samples and animal models. However, patient studies often miss the data from the crucial early stage of cardiovascular diseases, as obtaining primary tissues at this stage is impracticable. Transgenic animal models can offer some insights into disease mechanisms, although they usually do not fully recapitulate the phenotype of cardiovascular diseases and their progression. In recent years, a promising breakthrough has emerged in the form of in vitro three-dimensional (3D) cardiovascular models utilizing human pluripotent stem cells. These innovative models recreate the intricate 3D structure of the human heart and vessels within a controlled environment. This advancement is pivotal as it addresses the existing gaps in cardiovascular research, allowing scientists to study different stages of cardiovascular diseases and specific drug responses using human-origin models. In this review, we first outline various approaches employed to generate these models. We then comprehensively discuss their applications in studying cardiovascular diseases by providing insights into molecular and cellular changes associated with cardiovascular conditions. Moreover, we highlight the potential of these 3D models serving as a platform for drug testing to assess drug efficacy and safety. Despite their immense potential, challenges persist, particularly in maintaining the complex structure of 3D heart and vessel models and ensuring their function is comparable to real organs. However, overcoming these challenges could revolutionize cardiovascular research. It has the potential to offer comprehensive mechanistic insights into human-specific disease processes, ultimately expediting the development of personalized therapies.
... Furthermore, they have been designed the 'Biowire II' was designed to reconstruct cardiac tissue and CVDs modeling [145,[213][214][215]. Biowire II is an update of the previous version and the new generation can realize the separation or co-culture of atrial and ventricular tissues on a microfluidic device (figure 11(A)) and the device can also be used for heart disease modeling and disease gene screening. ...
Cardiovascular diseases (CVDs) are a major cause of death worldwide, leading to increased medical care costs. To turn the scale, it is essential to acquire a more in-depth and comprehensive understanding of CVDs and thus formulate more efficient and reliable treatments. Over the last decade, tremendous effort has been made to develop microfluidic systems to recapitulate native cardiovascular environments because of their unique advantages over conventional 2D culture systems and animal models such as high reproductivity, physiological relevance, and good controllability. These novel microfluidic systems could be extensively adopted for natural organ simulation, disease modeling, drug screening, disease diagnosis and therapy. Here, a brief review of the innovative designs of microfluidic devices for CVDs research is presented, with specific discussions on material selection, critical physiological and physical considerations. In addition, we elaborate on various biomedical applications of these microfluidic systems such as blood-vessel-on-a-chip and heart-on-a-chip, which are conducive to the investigation of the underlying mechanisms of CVDs. This review also provides systematic guidance on the construction of next-generation microfluidic systems for diagnosis and treatment of CVDs. Finally, the challenges and future directions in this field are highlighted and discussed.
... The currently available CM culturing devices in the market offer a number of essential stimuli and sensing for the physiological cultivation and maturation of hiPSC-CMs [310][311][312][313][314]. These engineered heart tissues and heart-on-a-chip devices can be supported by hydrogel substrates and equipped with flexible wires to record active force and passive tension, calcium transients and action potentials combined with contractility sensing, to generate 3Dengineered cardiac tissues from hiPSC-CMs, used subsequently for pharmacology studies [315]. ...
... Most groups have developed heart-on-a-chip platforms in which cardiomyocytes are integrated, sometimes combined with cardiac fibroblasts. (Mastikhina et al., 2020;Xiao et al., 2014;Ogle et al., 2016) Although these platforms have proven to be capable of mimicking many aspects of heart disease, including cardiac fibrosis, the use of multiple cell types has prohibited focusing on mechanotransduction in cardiac fibroblasts specifically. ...
In cardiac fibrosis, in response to stress or injury, cardiac fibroblasts deposit excessive amounts of collagens which contribute to the development of heart failure. The biochemical stimuli in this process have been extensively studied, but the influence of cyclic deformation on the fibrogenic behavior of cardiac fibroblasts in the ever-beating heart is not fully understood. In fact, most investigated mechanotransduction pathways in cardiac fibroblasts seem to ultimately have profibrotic effects, which leaves an important question in cardiac fibrosis research unanswered: how do cardiac fibroblasts stay quiescent in the ever-beating human heart? In this study, we developed a human cardiac fibrosis-on-a-chip platform and utilized it to investigate if and how cyclic strain affects fibrogenic signaling. The pneumatically actuated platform can expose engineered tissues to controlled strain magnitudes of 0-25% - which covers the entire physiological and pathological strain range in the human heart - and to biochemical stimuli and enables high-throughput screening of multiple samples. Microtissues of human fetal cardiac fibroblasts (hfCF) embedded in gelatin methacryloyl (GelMA) were 3D-cultured on this platform and exposed to strain conditions which mimic the healthy human heart. The results provide evidence of an antifibrotic effect of the applied strain conditions on cardiac fibroblast behavior, emphasizing the influence of biomechanical stimuli on the fibrogenic process and giving a detailed overview of the mechanosensitive pathways and genes involved, which can be used in the development of novel therapies against cardiac fibrosis.
... A microfabricated bioreactor was designed to mimic natural cardiac bundles in vitro using cardiac bio-wires [100]. Type I collagen was selected as the main gel matrix, which is one of the primary components of native myocardium. ...
... Type I collagen was selected as the main gel matrix, which is one of the primary components of native myocardium. The collagen-based cardiac biowires remained stable in the bioreactor for weeks, with the mechanical support provided by the suspended templates made of either silk suture or polytetrafluoroethylene (PTFE) microtubing [100]. Nashimoto and colleagues presented a microfluidic device made of PDMS and fibrin-collagen gel culturing 3D cellular spheroids with a vascular network [101]. ...
Organ-on-A-chip (OoAC) devices are miniaturized, functional, in vitro constructs that aim to recapitulate the in vivo physiology of an organ using different cell types and extracellular matrix, while maintaining the chemical and mechanical properties of the surrounding microenvironments. From an end-point perspective, the success of a microfluidic OoAC relies mainly on the type of biomaterial and the fabrication strategy employed. Certain biomaterials, such as PDMS (polydimethylsiloxane), are preferred over others due to their ease of fabrication and proven success in modelling complex organ systems. However, the inherent nature of human microtissues to respond differently to surrounding stimulations has led to the combination of biomaterials ranging from simple PDMS chips to 3D-printed polymers coated with natural and synthetic materials, including hydrogels. In addition, recent advances in 3D printing and bioprinting techniques have led to the powerful combination of utilizing these materials to develop microfluidic OoAC devices. In this narrative review, we evaluate the different materials used to fabricate microfluidic OoAC devices while outlining their pros and cons in different organ systems. A note on combining the advances made in additive manufacturing (AM) techniques for the microfabrication of these complex systems is also discussed.
... There are a very large variety of cardiac MPS, though all aim to culture cardiomyocytes in a way that provides a greater level of physiological relevance compared to traditional 2D cell culture techniques, with additional emphasis placed on scalability and throughput. Examples of increased physiological relevance include cardiomyocyte anisotropy to mimic myocardial structure (Schneider et al., 2019;López-Canosa et al., 2021), the inclusion of a microscale perfusable wire to mimic microvasculature ( Fig. 1F) (Xiao et al., 2014), the application of cyclic strain to cardiac tissues (Marsano et al., 2016), and the incorporation of multiple tissue and/or organ types to allow for paracrine signaling between different cell populations (Loskill et al., 2015). MPS can also be designed to allow for more precise control over drug perfusion rates by mimicking in vivo microvasculature ( Fig. 1G) (Mathur et al., 2015). ...
Heart disease is the leading cause of death worldwide and imposes a significant burden on healthcare systems globally. A major hurdle to the development of more effective therapeutics is the reliance on animal models that fail to faithfully recapitulate human pathophysiology. The predictivity of in vitro models that lack the complexity of in vivo tissue remain poor as well. To combat these issues, researchers are developing organ-on-a-chip models of the heart that leverage the use of human induced pluripotent stem cell-derived cardiomyocytes in combination with novel platforms engineered to better recapitulate tissue- and organ-level physiology. The integration of novel biosensors into these platforms is also a critical step in the development of these models, as they allow for increased throughput, real-time and longitudinal phenotypic assessment, and improved efficiency during preclinical disease modeling and drug screening studies. These platforms hold great promise for both improving our understanding of heart disease as well as for screening potential therapeutics based on clinically relevant endpoints with better predictivity of clinical outcomes. In this review, we describe state-of-the-art heart-on-a-chip platforms, the integration of novel biosensors into these models for real-time and continual monitoring of tissue-level physiology, as well as their use for modeling heart disease and drug screening applications. We also discuss future perspectives and further advances required to enable clinical trials-on-a-chip and next-generation precision medicine platforms.
... 3D static ''cardiac tissue mimetics'' composed of neonatal rat ventricular (NRMV) CMs and HUVECs were recently used to elucidate vasculogenic signaling mechanisms via characterization of endothelial network organization (Wagner et al., 2020). Meanwhile, other studies have created functional vascular structures, with both microfluidic and bioprinted models demonstrating perfusable myocardium with either acellular vascular channels (Xiao et al., 2014;Skylar-Scott et al., 2019) or a living endothelium (Sakaguchi et al., 2013;Zhang et al., 2016;Ellis et al., 2017;Noor et al., 2019). However, the approach demonstrated here couples beating CMs in direct contact with perfusable, living microvasculature while allowing cellular resolution microscopy for quantification of electrophysiology, such as calcium transients and contractility, and physiological phenomena, such as CM contractile contribution to blood flow. ...
In this study, we report static and perfused models of human myocardial-microvascular interaction. In static culture, we observe distinct regulation of electrophysiology of human induced pluripotent stem cell derived-cardiomyocytes (hiPSC-CMs) in co-culture with human cardiac microvascular endothelial cells (hCMVECs) and human left ventricular fibroblasts (hLVFBs), including modification of beating rate, action potential, calcium handling, and pro-arrhythmic substrate. Within a heart-on-a-chip model, we subject this three-dimensional (3D) co-culture to microfluidic perfusion and vasculogenic growth factors to induce spontaneous assembly of perfusable myocardial microvasculature. Live imaging of red blood cells within myocardial microvasculature reveals pulsatile flow generated by beating hiPSC-CMs. This study therefore demonstrates a functionally vascularized in vitro model of human myocardium with widespread potential applications in basic and translational research.
