Vratislav Cmiel

Phenotypic assays for analyses of pluripotent stem cell-derived cardiomyocytes

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.

Smartphone based point-of-care detector of urine albumin

Albumin plays an important role in human body. Its changed level in urine may indicate serious kidney disorders. We present a new point-of-care solution for sensitive detection of urine albumin - the miniature optical adapter for iPhone with in-built optical filters and a sample slot. The adapter exploits smart-phone flash to generate excitation light and camera to measure the level of emitted light. Albumin Blue 580 is used as albumin reagent. The proposed light-weight adapter can be produced at low cost using a 3D printer. Thus, the miniaturized detector is easy to use out of lab.

Computer analysis of isolated cardiomyocyte contraction process via advanced image processing techniques

Isolated cardiomyocytes have been used as valid and useful model in experimental cardiology research for decades. The cell contraction function is usually measured via expensive and complex instruments which can either damage the cell or take much time for setting up. In contrary, recent development of optical microscopy and digital cameras suggests utilization of touch-less cardiomyocyte video acquisition in connection with advanced image processing techniques for evaluation of the cell contraction process. The proposed paper presents an automatic membrane detection method via computer processing of acquired video-sequences by utilization of an active contour model. Evaluation of detected cell area is used for estimation of cardiomyocyte contraction function. The method is evaluated utilizing the comparison with contraction measurement performed via atomic force microscopy technique.

Method for adult cardiomyocyte long-term viability monitoring using confocal microscopy techniques

Freshly isolated myocytes lose their viability and functionality very early, from single hours to single days. So their viability is usually tested before the experiments or monitored continuously in periodic time intervals. But simple observations using viability kits (e. g. LIVE / DEAD Cell Imaging Kit, Life Technologies) for this purpose are not sufficient because of Calcein photobleaching and furthermore repeated testing is not possible. In this paper we tested advanced methods based on Calcium degradation and fluorescence lifetime measurement or cardiomyocyte shape properties calculations.

Real-time measurement of cardiomyocyte contraction and calcium transients using fast image processing algorithms

An improved technique for recording of contrac-tion of heart cells simultaneously with recording of calcium transients by fluorescence method is introduced. The tech-nique aims to quantify contractions under conditions including low image quality caused by reduced transmitted light. The proposed methods allow more accurate detection, lower error rate using the up-to-date image processing, and automation of the process comparing to recent approaches. The technique is compared with direct optical measurement method using processing of two-dimensional digital images of the cardiac cell. The technique is improved to be fast enough to be includ-ed in the system for real-time measurements.

Systems for rapid simultaneous measurement of calcium transients and contractions of adult cardiomyocytes

Two measurement setups with different optical adapters that enable multichannel rapid simultaneous measurement of calcium transients and length changes of adult cardiac cells are presented and compared on a sample experimental data taken from experiments.

Modern Semi-automatic Set-up for Testing Cell Migration with Impact for Therapy of Myocardial Infarction

Ischemic heart disease and resulting acute myocardial infarction (AMI) is one of the main causes of morbidity and mortality in industrial countries. The idea for the modern therapeutic strategy, which should activate the migration of stem/progenitor cells or reduce the migration of inflammatory cells in AMI regions, has emerged in the last 15 years, mainly as a result of physiological observation and post-mortem histology. Published data from direct measurements of cell migration are very limited. We prepared a universal set-up that can be used for the testing of cell migration in AMI micro-environment. Mesenchymal stromal cells (MSCs), the most commonly used stem/progenitor cells in experimental cellular therapy for AMI, were used in the recent set-up tests. The cells, which should be tested for their migration potential, were injected into the starting point in a special micro-chamber on the substrate, and optics of the microscope allowed a time-lapse recording of cells in micrometre resolution every 2 min. Our software tools provided precise 2D and 3D tracking of moving cells and data export for statistical analysis. Set-up should be upgraded to a fully-automatic preclinical screening tool in the future.

DNA Intracellular Delivery into 3T3 Cell Line Using Fluorescence Magnetic Ferumoxide Nanoparticles

Gene delivery is a widespread strategy in current experimental medicine. In this work, we report a method for low-toxic intracellular DNA vector delivery and post transfection localisation of this vector in mouse embryonic fibroblast cell lines. The surface of modified ferumoxide nanoparticles conjugated with Rhoda-mine B isothiocyanate (FeNV-Rh) was modified with linear polyethyleneimine and medium molecular weight chitosan to increase Accelerated Sensor of Action Potentials DNA vector adhesion. The size of the FeNV-Rh/DNA transfection complex was studied using dynamic light scattering (DLS) and scanning electron microscopy (SEM) techniques. The transfection complex internalisation of plasmid expression and FeNV-Rh, and stability of rhodamine fluorescence in intracellular space were observed at time periods 6, 12, 24 and 48 h post transfection. Results showed high transfection complex intracellular biocompatibility—cell viability after Rh-MNP labelling was higher than 97% 24 h after transfection, and higher than 95% after the next 24 h. Selective FeNV-Rh localisation in the lysosomes was quantified. More than 82% of nanoparticles were localised in the lysosomes 12 h post transfection and 94% of lysosomes had a significant and long-term deposit of nanoparticles. DNA vector expression was visible in >65% of the cells and precise protein localisation on the cell membrane was confirmed using confocal microscopy.

Simultaneous study of mechanobiology and calcium dynamics on hESC-derived cardiomyocytes clusters

Calcium ions act like ubiquitous second messengers in a wide amount of cellular processes. In cardiac myocytes, Ca2+ handling regulates the mechanical contraction necessary to the heart pump function. The field of intracellular and intercellular Ca2+ 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.

Three-dimensional fluorescence lifetime imaging in confocal microscopy of living cells

Fluorescence lifetime imaging (FLIM) is a modern optical method which increases the potential of standard microscopy. This paper shows the possibilities of extended fluorescence lifetime evaluation and imaging in studying three-dimensional structures such as compartments of living cells with different fluorescence lifetimes. The method for quasi-FLIM image calculation is presented and image processing steps useful for biological experiments are suggested. The method was tested on isolated cardiomyocyte cells (CMs) and rat bone marrow stromal cells (MSCs) labelled with SPIO-rhodamine nanoparticles and stained with standard fluorescent dyes. We proved it is possible to use an exponential decrease of fluorescence in time and lifetime parameters for pseudo-colour 3D image mapping of living cells and their compartments that is not a standard function of confocal microscopes.