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Stem cell-derived cardiomyocytes (CMs) hold great hopes for myocardium regeneration because of their ability to produce functional cardiac cells in large quantities. They also hold promise in dissecting the molecular principles involved in heart diseases and also in drug development, owing to their ability to model the diseases using patient-specific human pluripotent stem cell (hPSC)-derived CMs. The CM properties essential for the desired applications are frequently evaluated through morphologic and genotypic screenings. Even though these characterizations are necessary, they cannot in principle guarantee the CM functionality and their drug response. The CM functional characteristics can be quantified by phenotype assays, including electrophysiological, optical, and/or mechanical approaches implemented in the past decades, especially when used to investigate responses of the CMs to known stimuli (eg, adrenergic stimulation). Such methods can be used to indirectly determine the electrochemomechanics of the cardiac excitation-contraction coupling, which determines important functional properties of the hPSC-derived CMs, such as their differentiation efficacy, their maturation level, and their functionality. In this work, we aim to systematically review the techniques and methodologies implemented in the phenotype characterization of hPSC-derived CMs. Further, we introduce a novel approach combining atomic force microscopy, fluorescent microscopy, and external electrophysiology through microelectrode arrays. We demonstrate that this novel method can be used to gain unique information on the complex excitation-contraction coupling dynamics of the hPSC-derived CMs.

Alignment and patterning of neurons have great importance in some research fields, especially for regenerative medicine and for the formation of artificial neural networks. Alignment of neurons on quartz grooves and parylene cues on quartz substrate was evaluated in this work. The neurons’ alignment on quartz grooves is considered to be topographical alignment, while the neurons’ alignment on parylene cues on quartz substrate is considered to be chemical alignment. Both quartz grooves’ and parylene cues’ widths were fabricated in a range from 2 µm to 8 µm; quartz grooves’ heights were in a range from 0.25 µm to 4 µm, while parylene cues’ heights were only 0.25 µm. Neurons were dissociated hippocampal neurons from rat E18. Neurons were cultivated on test substrates for 7 days before alignment evaluation. As expected, neurons aligned according to the direction of grooves and cues; however, transversal growth direction was also observed with much smaller tendency. Chemical alignment was found to be more effective than topographical alignment. If parylene cues are thin and distanced enough, then neurons have a tendency to follow the direction of individual parylene cues; however, neurons on quartz grooves have a tendency just to follow a preferable direction than individual quartz grooves.

Calcium ions act like ubiquitous second messengers in a wide amount of cellular processes. In cardiac myocytes, Ca handling regulates the mechanical contraction necessary to the heart pump function. The field of intracellular and intercellular Ca handling, employing in vitro models of cardiomyocytes, has become a cornerstone to understand the role and adaptation of calcium signalling in healthy and diseased hearts. Comprehensive in vitro systems and cell-based biosensors are powerful tools to enrich and speed up cardiac phenotypic and drug response evaluation. We have implemented a combined setup to measure contractility and calcium waves in human embryonic stem cells-derived cardiomyocyte 3D clusters, obtained from embryoid body differentiation. A combination of atomic force microscopy to monitor cardiac contractility, and sensitive fast scientific complementary metal-oxide-semiconductor camera for epifluorescence video recording, provided correlated signals in real time. To speed up the integrated data processing, we tested several post-processing algorithms, to improve the automatic detection of relevant functional parameters. The validation of our proposed method was assessed by caffeine stimulation (10mM) and detection/characterization of the induced cardiac response. We successfully report the first simultaneous recording of cardiac contractility and calcium waves on the described cardiac 3D models. The drug stimulation confirmed the automatic detection capabilities of the used algorithms, measuring expected physiological response, such as elongation of contraction time and Ca cytosolic persistence, increased calcium basal fluorescence, and transient peaks. These results contribute to the implementation of novel, integrated, high-information, and reliable experimental systems for cardiac models and drug evaluation.

In the last few years, magnetically labeled cells have been intensively explored, and non-invasive cell tracking and magnetic manipulation methods have been tested in preclinical studies focused on cell transplantation. For clinical applications, it is desirable to know the intracellular pathway of nanoparticles, which can predict their biocompatibility with cells and the long-term imaging properties of labeled cells. Here, we quantified labeling efficiency, localization, and fluorescence properties of Rhodamine derivatized superparamagnetic maghemite nanoparticles (SAMN-R) in mesenchymal stromal cells (MSC). We investigated the stability of SAMN-R in the intracellular space during a long culture (20 days). Analyses were based on advanced confocal microscopy accompanied by atomic absorption spectroscopy (AAS) and magnetic resonance imaging. SAMN-R displayed excellent cellular uptake (24 h of labeling), and no toxicity of SAMN-R labeling was found. 83% of SAMN-R nanoparticles were localized in lysosomes, only 4.8% were found in mitochondria, and no particles were localized in the nucleus. On the basis of the MSC fluorescence measurement every 6 days, we also quantified the continual decrease of SAMN-R fluorescence in the average single MSC during 18 days. An additional set of analyses showed that the intracellular SAMN-R signal decrease was minimally caused by fluorophore degradation or nanoparticles extraction from the cells, main reason is a cell division. The fluorescence of SAMN-R nanoparticles within the cells was detectable minimally for 20 days. These observations indicate that SAMN-R nanoparticles have a potential for application in transplantation medicine.

It has been shown that, in addition to conventional contact electrode techniques, optical methods using fluorescent dyes can be successfully used for cardiac signal measurement. In this review, the physical and technical fundamentals of the method are described, as well as the properties of the most common systems for measuring action potentials and intracellular calcium concentration. Special attention is paid to summarizing limitations and trends in developing this method.

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