Gellan gum-gelatin based cardiac models support formation of cellular networks and functional cardiomyocytes.

Cardiovascular diseases remain as the most common cause of death worldwide. To reveal the underlying mechanisms in varying cardiovascular diseases, in vitro models with cells and supportive biomaterial can be designed to recapitulate the essential components of human heart. In this study, we analyzed whether 3D co-culture of cardiomyocytes (CM) with vascular network and with adipose tissue-derived mesenchymal stem/stromal cells (ASC) can support CM functionality. CM were cultured with either endothelial cells (EC) and ASC or with only ASC in hydrazide-modified gelatin and oxidized gellan gum hybrid hydrogel to form cardiovascular multiculture and myocardial co-culture, respectively. We studied functional characteristics of CM in two different cellular set-ups and analyzed vascular network formation, cellular morphology and orientation. The results showed that gellan gum-gelatin hydrogel supports formation of two different cellular networks and functional CM. We detected formation of a modest vascular network in cardiovascular multiculture and extensive ASC-derived alpha smooth muscle actin -positive cellular network in multi- and co-culture. iPSC-CM showed elongated morphology, partly aligned orientation with the formed networks and presented normal calcium transients, beating rates, and contraction and relaxation behavior in both setups. These 3D cardiac models provide promising platforms to study (patho) physiological mechanisms of cardiovascular diseases. Supplementary Information: The online version contains supplementary material available at 10.1007/s10616-024-00630-5.

Multifocal optical projection microscopy enables label-free 3D measurement of cardiomyocyte cluster contractility.

Human induced pluripotent stem cell (hiPSC)-derived cardiomyocyte (CM) models have become an attractive tool for in vitro cardiac disease modeling and drug studies. These models are moving towards more complex three-dimensional microphysiological organ-on-chip systems. Label-free imaging-based techniques capable of quantifying contractility in 3D are needed, as traditional two-dimensional methods are ill-suited for 3D applications. Here, we developed multifocal (MF) optical projection microscopy (OPM) by integrating an electrically tunable lens to our in-house built optical projection tomography setup for extended depth of field brightfield imaging in CM clusters. We quantified cluster biomechanics by implementing our previously developed optical flow-based CM video analysis for MF-OPM. To demonstrate, we acquired and analyzed multiangle and multifocal projection videos of beating hiPSC-CM clusters in 3D hydrogel. We further quantified cluster contractility response to temperature and adrenaline and observed changes to beating rate and relaxation. Challenges emerge from light penetration and overlaying textures in larger clusters. However, our findings indicate that MF-OPM is suitable for contractility studies of 3D clusters. Thus, for the first time, MF-OPM is used in CM studies and hiPSC-CM 3D cluster contraction is quantified in multiple orientations and imaging planes.

Building blocks of microphysiological system to model physiology and pathophysiology of human heart.

Microphysiological systems (MPS) are drawing increasing interest from academia and from biomedical industry due to their improved capability to capture human physiology. MPS offer an advanced in vitro platform that can be used to study human organ and tissue level functions in health and in diseased states more accurately than traditional single cell cultures or even animal models. Key features in MPS include microenvironmental control and monitoring as well as high biological complexity of the target tissue. To reach these qualities, cross-disciplinary collaboration from multiple fields of science is required to build MPS. Here, we review different areas of expertise and describe essential building blocks of heart MPS including relevant cardiac cell types, supporting matrix, mechanical stimulation, functional measurements, and computational modelling. The review presents current methods in cardiac MPS and provides insights for future MPS development with improved recapitulation of human physiology.

A Portable Microscale Cell Culture System with Indirect Temperature Control.

A physiologically relevant environment is essential for successful long-term cell culturing in vitro. Precise control of temperature, one of the most crucial environmental parameters in cell cultures, increases the fidelity and repeatability of the experiments. Unfortunately, direct temperature measurement can interfere with the cultures or prevent imaging of the cells. Furthermore, the assessment of dynamic temperature variations in the cell culture area is challenging with the methods traditionally used for measuring temperature in cell culture systems. To overcome these challenges, we integrated a microscale cell culture environment together with live-cell imaging and a precise local temperature control that is based on an indirect measurement. The control method uses a remote temperature measurement and a mathematical model for estimating temperature at the desired area. The system maintained the temperature at 37±0.3 °C for more than 4 days. We also showed that the system precisely controls the culture temperature during temperature transients and compensates for the disturbance when changing the cell cultivation medium, and presented the portability of the heating system. Finally, we demonstrated a successful long-term culturing of human induced stem cell-derived beating cardiomyocytes, and analyzed their beating rates at different temperatures.

Evaluation of Optogenetic Electrophysiology Tools in Human Stem Cell-Derived Cardiomyocytes.

Current cardiac drug safety assessments focus on hERG channel block and QT prolongation for evaluating arrhythmic risks, whereas the optogenetic approach focuses on the action potential (AP) waveform generated by a monolayer of human cardiomyocytes beating synchronously, thus assessing the contribution of several ion channels on the overall drug effect. This novel tool provides arrhythmogenic sensitizing by light-induced pacing in combination with non-invasive, all-optical measurements of cardiomyocyte APs and will improve assessment of drug-induced electrophysiological aberrancies. With the help of patch clamp electrophysiology measurements, we aimed to investigate whether the optogenetic modifications alter human cardiomyocytes' electrophysiology and how well the optogenetic analyses perform against this gold standard. Patch clamp electrophysiology measurements of non-transduced stem cell-derived cardiomyocytes compared to cells expressing the commercially available optogenetic constructs Optopatch and CaViar revealed no significant changes in action potential duration (APD) parameters. Thus, inserting the optogenetic constructs into cardiomyocytes does not significantly affect the cardiomyocyte's electrophysiological properties. When comparing the two methods against each other (patch clamp vs. optogenetic imaging) we found no significant differences in APD parameters for the Optopatch transduced cells, whereas the CaViar transduced cells exhibited modest increases in APD-values measured with optogenetic imaging. Thus, to broaden the screen, we combined optogenetic measurements of membrane potential and calcium transients with contractile motion measured by video motion tracking. Furthermore, to assess how optogenetic measurements can predict changes in membrane potential, or early afterdepolarizations (EADs), cells were exposed to cumulating doses of E-4031, a hERG potassium channel blocker, and drug effects were measured at both spontaneous and paced beating rates (1, 2 Hz). Cumulating doses of E-4031 produced prolonged APDs, followed by EADs and drug-induced quiescence. These observations were corroborated by patch clamp and contractility measurements. Similar responses, although more modest were seen with the IKs potassium channel blocker JNJ-303. In conclusion, optogenetic measurements of AP waveforms combined with optical pacing compare well with the patch clamp gold standard. Combined with video motion contractile measurements, optogenetic imaging provides an appealing alternative for electrophysiological screening of human cardiomyocyte responses in pharmacological efficacy and safety testings.

Methods for in vitro functional analysis of iPSC derived cardiomyocytes - Special focus on analyzing the mechanical beating behavior.

A rapidly increasing number of papers describing novel iPSC models for cardiac diseases are being published. To be able to understand the disease mechanisms in more detail, we should also take the full advantage of the various methods for analyzing these cell models. The traditionally and commonly used electrophysiological analysis methods have been recently accompanied by novel approaches for analyzing the mechanical beatingbehavior of the cardiomyocytes. In this review, we provide first a concise overview on the methodology for cardiomyocyte functional analysis and then concentrate on the video microscopy, which provides a promise for a new faster yet reliable method for cardiomyocyte functional analysis. We also show how analysis conditions may affect the results. Development of the methodology not only serves the basic research on the disease models, but could also provide the much needed efficient early phase screening method for cardiac safety toxicology. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Integration of Developmental and Environmental Cues in the Heart edited by Marcus Schaub and Hughes Abriel.

Distinct electrophysiological and mechanical beating phenotypes of long QT syndrome type 1-specific cardiomyocytes carrying different mutations.

BACKGROUND: Long QT syndrome (LQTS) is associated with increased risk of ventricular arrhythmias and cardiac arrest. LQTS type 1 (LQT1), the most prevalent subtype of LQTS, is caused by defects of slow delayed rectifier potassium current (IKs) that lead to abnormal cardiac repolarization. Here we used pluripotent stem cell (iPSC)-technology to investigate both the electrophysiological and also for the first time the mechanical beating behavior of genetically defined, LQT1 specific cardiomyocytes (CMs) carrying different mutations. METHODS: We established in vitro models for LQT1 caused by two mutations (G589D or ivs7-2A>G). LQT1 specific CMs were derived from patient specific iPSCs and characterized for their electrophysiology using a current clamp and Ca2 +-imaging. Their mechanical beating characteristics were analyzed with video-image analysis method. RESULTS AND CONCLUSIONS: Both LQT1-CM-types showed prolonged repolarization, but only those with G589D presented early after-depolarizations at baseline. Increased amounts of abnormal Ca2 + transients were detected in both types of LQT1-CMs. Surprisingly, also the mechanical beating behavior demonstrated clear abnormalities and additionally the abnormalities were different with the two mutations: prolonged contraction was seen in G589D-CMs while impaired relaxation was observed in ivs7-2A>G-CMs. The CMs carrying two different LQT1 specific mutations (G589D or ivs7-2A>G) presented clear differences in their electrical properties as well as in their mechanical beating behavior. Results from different methods correlated well with each other suggesting that simply mechanical beating behavior of CMs could be used for screening of diseased CMs and possibly for diagnostic purposes in the future.

Video image-based analysis of single human induced pluripotent stem cell derived cardiomyocyte beating dynamics using digital image correlation.

BACKGROUND: The functionality of a cardiomyocyte is primarily measured by analyzing the electrophysiological properties of the cell. The analysis of the beating behavior of single cardiomyocytes, especially ones derived from stem cells, is challenging but well warranted. In this study, a video-based method that is non-invasive and label-free is introduced and applied for the study of single human cardiomyocytes derived from induced pluripotent stem cells. METHODS: The beating of dissociated stem cell-derived cardiomyocytes was visualized with a microscope and the motion was video-recorded. Minimum quadratic difference, a digital image correlation method, was used for beating analysis with geometrical sectorial cell division and radial/tangential directions. The time series of the temporal displacement vector fields of a single cardiomyocyte was computed from video data. The vector field data was processed to obtain cell-specific, contraction-relaxation dynamics signals. Simulated cardiomyocyte beating was used as a reference and the current clamp of real cardiomyocytes was used to analyze the electrical functionality of the beating cardiomyocytes. RESULTS: Our results demonstrate that our sectorized image correlation method is capable of extracting single cell beating characteristics from the video data of induced pluripotent stem cell-derived cardiomyocytes that have no clear movement axis, and that the method can accurately identify beating phases and time parameters. CONCLUSION: Our video analysis of the beating motion of single human cardiomyocytes provides a robust, non-invasive and label-free method to analyze the mechanobiological functionality of cardiomyocytes derived from induced pluripotent stem cells. Thus, our method has potential for the high-throughput analysis of cardiomyocyte functions.

Electrical Field Stimulation with a Novel Platform: Effect on Cardiomyocyte Gene Expression but not on Orientation.

Electrical field stimulation has been shown to improve cardiac cell alignment and functional properties. In this study, neonatal rat cardiomyocytes were exposed to both long-term and short-term stimulation with the goal of investigating whether it is possible to achieve cell orientation and the maturation of cardiomyocytes with a novel, microelectrode array (MEA)-compatible electrical stimulation platform. Cells were viable after electrical stimulation, but no orientation or other morphological changes were observed. However, the electrode wires in MEA dishes affected the cell orientation. Cell contractions synchronized with pacing, but settled back to their original frequency in the absence of stimulation. The expression of genes encoding a gap junction protein connexin-43 (Cx-43), and contractile cardiac protein beta myosin heavy chain 7, was stronger in stimulated cells than in controls (p<0.05). In summary, the surface topography influenced to cardiomyocyte orientation, suggesting that the micro architecture of the biomaterials should be carefully designed for cell applications. However, as electrical stimulation and its duration affected gene expression of some main cardiac proteins, the stimulation system may prove useful to enhance the cardiac differentiation of stem cells.