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Functional properties of cardiac microtissues. (A) Excitation threshold determined at the end of cultivation as a minimum voltage required to induce synchronous contraction. (B) Maximum capture rate determined at the end of cultivation as the maximum tissue beating frequency. (C) Force of contraction. Control-cardiac microtissues cultivated in the PDMS wells without electrical or mechanical stimulation. 1 Hz cardiac microtissues cultivated in the PDMS wells in the presence of electrical field stimulation at 1 Hz 5% cardiac mictotissues cultivated in the PDMS stretched at 5% static strain without electrical stimulation. 5% strain +1 Hz cardiac microtissues stretched at 5% static strain and concurrently subjected to electrical field stimulation at 1 Hz. * denotes statistical significant by two-way ANOVA between 5% and 5% +1 Hz groups at specific pacing frequencies. Data represented as average ± standard deviation, N = 3.
Source publication
We describe here a bioreactor capable of applying electrical field stimulation in conjunction with static strain and on-line force of contraction measurements. It consisted of a polydimethylsiloxane (PDMS) tissue chamber and a pneumatically driven stretch platform. The chamber contained eight tissue microwells (8.05 mm in length and 2.5 mm in width...
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Citations
... Mechanical stretch and electrical stimuli, which increase during development, are critical regulators of gap junction formation and maintenance [152,153]. Notably, electrical stimulation has been shown to strongly enhance many hallmarks of maturation in an in vitro cardiac tissue model, including the formation and maturation of ICD structures [154][155][156][157][158]. Mice lacking Cx43 exhibit abnormal cardiac conduction and increased susceptibility to arrhythmias, underscoring the importance of gap junction integrity and Cx43-mediated electrical coupling in cardiac maturation and function [159,160]. ...
Purpose of the Review
This review aims to discuss the process of cardiomyocyte maturation, with a focus on the underlying molecular mechanisms required to form a fully functional heart. We examine both long-standing concepts associated with cardiac maturation and recent developments, and the overall complexity of molecularly integrating all the processes that lead to a mature heart.
Recent Findings
Cardiac maturation, defined here as the sequential changes that occurring before the heart reaches full maturity, has been a subject of investigation for decades. Recently, there has been a renewed, highly focused interest in this process, driven by clinically motivated research areas where enhancing maturation may lead to improved therapeutic opportunities. These include using pluripotent stem cell models for cell therapy and disease modeling, as well as recent advancements in adult cardiac regeneration approaches.
Summary
We highlight key processes underlying maturation of the heart, including cellular and organ growth, and electrophysiological, metabolic, and contractile maturation. We further discuss how these processes integrate and interact to contribute to the overall complexity of the developing heart. Finally, we emphasize the transformative potential for translating relevant maturation concepts to emerging models of heart disease and regeneration.
... Other methods include prolonging culture time [10,11], co-culture with different cell types [12], culturing iPSC-CMs on parallel microstructures [13][14][15], using substrates with myocardial stiffness [16], conductive extracellular matrices [17], electrical stimulation [13,18] and small chemical molecules [19]. It seems that the combination of two or more methods to treat iPSC-CMs can achieve better results [20][21][22].Therefore, it is necessary to integrate several techniques and develop new platforms to improve the maturation of iPSC-CMs and achieve a phenotype closer to that of native adult cardiomyocytes. This will help to obtain data that more closely resembles that of the adult. ...
... In many myocardial diseases, deformation and remodelling of the myocardial interstitial network occurs, affecting myocardial contractile function and perfusion [24]. Furthermore, cell alignment has been shown to be one of the most important parameters affecting the function of engineered cardiac tissue [21,[25][26][27][28][29]. Motlagh [30] has posited that surface topography have an impact on cardiomyocyte shape, gene expression and protein distribution. ...
The preclinical evaluation of drug-induced cardiotoxicity is critical for developing novel drug, helping to avoid drug wastage and post-marketing withdrawal. Although human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) and the engineered heart organoid have been used for drug screening and mimicking disease models, they are always limited by the immaturity and lack of functionality of the cardiomyocytes. In this study, we constructed a Cardiomyocytes-on-a-Chip (CoC) that combines micro-grooves (MGs) and circulating mechanical stimulation to recapitulate the well-organized structure and stable beating of myocardial tissue. The phenotypic changes and maturation of CMs cultured on the CoC have been verified and can be used for the evaluation of cardiotoxicity and cardioprotective drug responses. Taken together, these results highlight the ability of our myocardial microarray platform to accurately reflect clinical behaviour, underscoring its potential as a powerful pre-clinical tool for assessing drug response and toxicity.
... The use of a device where cells are challenged just by mechanical stimuli oversimplifies the real physiological situation where other stimuli, such as chemical diffusion of paracrine molecules, shear stress or interstitial fluid flow might be present. So it would be important to consider experiments at tissue level, where different types of cells, such as for example cardiac fibroblasts and cardiomyocytes, are exposed to different stimuli [171,172]. If seeing is believing, the integration of live imaging based on high time and spatial resolutions with cell stretchers further improves the potentiality of these approaches. ...
Mechanical stimuli have multiple effects on cell behavior, affecting a number of cellular processes including orientation, proliferation or apoptosis, migration and invasion, the production of extracellular matrix proteins, the activation and translocation of transcription factors, the expression of different genes such as those involved in inflammation and the reprogramming of cell fate. The recent development of cell stretching devices has paved the way for the study of cell reactions to stretching stimuli in-vitro, reproducing physiological situations that are experienced by cells in many tissues and related to functions such as breathing, heart beating and digestion. In this work, we review the highly-relevant contributions cell stretching devices can provide in the field of mechanobiology. We then provide the details for the in-house construction and operation of these devices, starting from the systems that we already developed and tested. We also review some examples where cell stretchers can supply meaningful insights into mechanobiology topics and we introduce new results from our exploitation of these devices.
... While these strategies have usually proven successful in improving Cx43 expression, the increase is typically modest at best. Another intervention that appears to reliably influence Cx43 expression and its proper subcellular localization is the application of static or cyclic stretch [71][72][73]. In an early work, Salameh and colleagues (2010) found that cyclic mechanical stretch induced cellular elongation and increased the expression and anisotropy of Cx43 GJs in rat neonatal cardiomyocytes [73]. ...
The transplantation of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) has shown promise in preclinical models of myocardial infarction, but graft myocardium exhibits incomplete host–graft electromechanical integration and a propensity for pro-arrhythmic behavior. Perhaps contributing to this situation, hPSC-CM grafts show low expression of connexin 43 (Cx43), the major gap junction (GJ) protein, in ventricular myocardia. We hypothesized that Cx43 expression and function could be rescued by engineering Cx43 in hPSC-CMs with a series of phosphatase-resistant mutations at three casein kinase 1 phosphorylation sites (Cx43-S3E) that have been previously reported to stabilize Cx43 GJs and reduce arrhythmias in transgenic mice. However, contrary to our predictions, transgenic Cx43-S3E hPSC-CMs exhibited reduced Cx43 expression relative to wild-type cells, both at baseline and following ischemic challenge. Cx43-S3E hPSC-CMs showed correspondingly slower conduction velocities, increased automaticity, and differential expression of other connexin isoforms and various genes involved in cardiac excitation–contraction coupling. Cx43-S3E hPSC-CMs also had phosphorylation marks associated with Cx43 GJ internalization, a finding that may account for their impaired GJ localization. Taken collectively, our data indicate that the Cx43-S3E mutation behaves differently in hPSC-CMs than in adult mouse ventricular myocytes and that multiple biological factors likely need to be addressed synchronously to ensure proper Cx43 expression, localization, and function.
... Using a rat model of a complete heart block, they implanted this strip outside of the heart, restoring atrioventricular conduction. Miklas et al. [18] prepared collagen gels embedded with neonatal rat heart-derived cardiomyocytes performing electrical and mechanical stimulation. Additionally, Nunes et al. [19] created self-assembled electrically stimulated cardiac biowires using iPSC-CMs. ...
Atrioventricular block (AVB) is a severe disease for pediatric patients. The repetitive operations needed in the case of the pacemaker implantation to maintain the electrical signal at the atrioventricular node (AVN) affect the patient’s life quality. In this study, we present a method of biofabrication of multi-cell-laden cylindrical fibrin-based fibers that can restore the electrical signal at the AVN. We used human umbilical vein smooth muscle cells (HUVSMCs), human umbilical vein endothelial cells (HUVECs) and induced pluripotent stem cell cardiomyocytes (iPSC-CMs) cultivated either statically or dynamically to mimic the native AVN. We investigated the influence of cell composition, construct diameter and cyclic stretch on the function of the fibrin hydrogels in vitro. Immunohistochemistry analyses showed the maturity of the iPSC-CMs in the constructs through the expression of sarcomeric alpha actinin (SAA) and electrical coupling through Connexin 43 (Cx43) signal. Simultaneously, the beating frequency of the fibrin hydrogels was higher and easy to maintain whereas the concentration of iPSC-CMs was higher compared with the other types of cylindrical constructs. In total, our study highlights that the combination of fibrin with the cell mixture and geometry is offering a feasible biofabrication method for tissue engineering approaches for the treatment of AVB.
... As described above, mechanical or electrical stimulation can individually promote the growth of cardiomyocytes cultured in vitro [8,38,39], but there are few studies [40][41][42][43] on the combined mechanical-electrical stimulation. There is an inseparable connection between the mechanical and electrical properties of cardiomyocytes, and the two can be transformed into each other, so it is required to keep cardiomyocytes in a force/electricity coupled environment [8,44]. ...
Engineered myocardial tissue is expected to be used in the treatment of myocardial defects and other diseases, and one of the keys is to construct a suitable environment for the culture of myocardial tissue in vitro. In this study, flow shear stress and pulse electrical stimulation were applied to cardiomyocytes with a self-designed device by simulating the mechanical and electrical physiological microenvironment of myocardial tissue. The strength and duration of pulse electrical stimulation as well as the intensity of shear stress were studied in detail to optimize the experimental parameters. Concretely, 100 mV pulse electrical stimulation (1 Hz and 10 ms pulse width) and 10 dyn/cm2 shear stress were used for studying the influence of combined mechanical-electrical stimulation to the growth of cardiomyocytes. The mechanical factor of the combined stimulation promoted the expression of -cardiac actin mRNA, the electrical factor caused an increase in Cx-43 mRNA expression, and shear stress and pulse electrical stimulation showed a synergistic action on the expression of GATA-4 mRNA. It indicated that combined mechanical-electrical stimulation had a better effect on the functionalized culture of cardiomyocytes, which provided an important theoretical basis for the further construction of in vitro engineered myocardial tissue.
... [21e,22] Further, remodeling and elongation of sarcomeric structures were observed when an uniaxial static strain was applied to CMs. [21d,23] On similar lines, the application of electromechanical stimulation (in terms of static stretch and external electrical stimula-tion) to CMs resulted in improved sarcomere structure with elevated contraction frequencies. [24] Native CTs generally experience a peak systolic strain of 5% to 20% depending on the heart conditions, age, and pressure load. Most of the fabricated bioreactors/microdevices are capable of providing these strains at 1-3 Hz cyclic stretch frequency and have reported both phenotypic and genotypic changes in CMs. ...
In vitro cardiomyocyte (CM) maturation is an imperative step to replicate native heart tissue‐like structures as cardiac tissue grafts or as drug screening platforms. CMs are known to interpret biophysical cues such as stiffness, topography, external mechanical stimulation or dynamic perfusion load through mechanotransduction and change their behavior, organization, and maturation. In this regard, a silk‐based cardiac tissue (CT) coupled with a dynamic perfusion‐based mechanical stimulation platform (DMM) for achieving maturation and functionality in vitro is tried to be delivered. Silk fibroin (SF) is used to fabricate lamellar scaffolds to provide native tissue‐like anisotropic architecture and is found to be nonimmunogenic and biocompatible allowing cardiomyocyte attachment and growth in vitro. Further, the scaffolds display excellent mechanical properties by their ability to undergo cyclic compressions without any deformation when places in the DMM. Gradient compression strains (5% to 20%), mimicking the native physiological and pathological conditions, are applied to the cardiomyocyte culture seeded on lamellar silk scaffolds in the DMM. A strain‐dependent difference in cardiomyocyte maturation, gene expression, sarcomere elongation, and extracellular matrix formation is observed. These silk‐based CTs matured in the DMM can open up several avenues toward the development of host‐specific grafts and in vitro models for drug screening.
... 55 In addition, an overall improvement in cell distribution and morphology, cardiac protein expression, and tissue organization is seen with the use of perfusion in cardiac cell aggregates. [56][57][58] Aggregates can also be molded into specific matrices or hydrogel to mimic the extracellular matrix environment of cardiac cells. 59,60 Crosslinked 3D networks composed of hydrophilic polymers mimic the soft and flexible structures and water content of native tissues, which promote a more physiologically accurate cardiac model. ...
Recent developments in applied developmental physiology have provided well-defined methodologies for producing human stem cell derived cardiomyocytes. The cardiomyocytes produced have become commonplace as cardiac physiology research models. Accessibility has also allowed for the development of tissue engineered human heart constructs for drug screening, surgical intervention, and investigating cardiac pathogenesis. However, cardiac tissue engineering is an interdisciplinary field that involves complex engineering and physiological concepts, which limits its accessibility. Our review provides a readable, broad reaching, and thorough discussion of major factors to consider for the development of cardiovascular tissues from stem cell derived cardiomyocytes. In this study, our review will examine important considerations in undertaking a cardiovascular tissue engineering project and will present, interpret, and summarize some of the recent advancements in this field. Throughout, we review different forms of tissue engineered constructs, a discussion on cardiomyocyte sources, and an in-depth discussion of the fabrication and maturation procedures for tissue engineered heart constructs.
Impact statement
With advancements in cardiac differentiation protocols, the production of human induced pluripotent stem cell derived cardiomyocytes is becoming cost effective and routine in the laboratory setting. Monolayer based culture methods are rapidly being replaced by three-dimensional (3D) tissue engineered constructs, which are more representative of the heart geometry. In the review presented, we delve into important concepts and tissue engineering principles that should be considered when generating 3D cardiac constructs, interpreting data acquired from, and embarking on a 3D cardiac tissue-based research project.
... For example, constraining the boundary conditions of a simple tissue construct promotes the evolution of a prescribed vessel network topology once implanted [4]. Similarly, stem cells that have been expanded, partially committed down a cardiomyocyte lineage enabling assembly into a tissue form and finally matured into the terminally differentiated cardiomyocyte following electrical and/or mechanical conditioning [5,6]. ...
Combining the power of biology with technology innovation to realize the promise of tissue repair by bioprinting regenerative tissues directly in a patient.
... The custom-designed elastic micropillars (termed as microfabricated tissue gauges) are not only used to constrain the engineered microtissues but also to report forces generated by them in real time. Such approach has attracted great interests in the past decade (Jason et al., 2014;Liu et al., 2014;Mills et al., 2017;Ronaldson-Bouchard et al., 2018;Chen and Zhao, 2019). Recently, a similar approach has been reported by using elastic microwires instead of micropillars to constrain and monitor microtissues, showing great promises in heteropolar microtissue construction and disease modeling (Mastikhina et al., 2019;Wang E. Y. et al., 2019;Zhao Y. et al., 2019). ...
Mechanical stretch is widely experienced by cells of different tissues in the human body and plays critical roles in regulating their behaviors. Numerous studies have been devoted to investigating the responses of cells to mechanical stretch, providing us with fruitful findings. However, these findings have been mostly observed from two-dimensional studies and increasing evidence suggests that cells in three dimensions may behave more closely to their in vivo behaviors. While significant efforts and progresses have been made in the engineering of biomaterials and approaches for mechanical stretching of cells in three dimensions, much work remains to be done. Here, we briefly review the state-of-the-art researches in this area, with focus on discussing biomaterial considerations and stretching approaches. We envision that with the development of advanced biomaterials, actuators and microengineering technologies, more versatile and predictive three-dimensional cell stretching models would be available soon for extensive applications in such fields as mechanobiology, tissue engineering, and drug screening.
