Konstantin Agladze

Electrospun nanofibers as a tool for architecture control in engineered cardiac tissue

This paper presents an in vitro system for cardiac tissue engineering based on cardiomyocytes cultured on electrospun polymethylglutarimide (PMGI) nanofibrous meshes either imprinted on solid substrate or suspended in space. Special care was taken over the ability to control the tissue architecture. The electrospinning process allowed nano-scale diameter PMGI fibers with different positioning density to be collected in a random or in an aligned way that defines the general configuration of the mesh. Micro-imprinted on solid substrate nanofibers guarantee aligned cell growth, when the distance between them is 30 μm or less. Suspended in 3D space, nanofibers define the overall architecture of the tissue, depending on orientation and positioning density of the nanofibers. As a result, cardiac cells proliferated into contractile tissue filaments, open-worked tissue meshes and continuous anisotropic cell sheets. Alignment of the cells was characterized by elongation of the cell shape and orientation of the α-actin filaments supported by the FFT data. The advantage of this method is its ability to maintain both three-dimensionality and structural anisotropy.

Curvature-dependent excitation propagation in cultured cardiac tissue

The geometry of excitation wave front may play an important role on the propagation block and spiral wave formation. The wave front which is bent over the critical value due to interaction with the obstacles may partially cease to propagate and appearing wave breaks evolve into rotating waves or reentry. This scenario may explain how reentry spontaneously originates in a heart. We studied highly curved excitation wave fronts in the cardiac tissue culture and found that in the conditions of normal, non-inhibited excitability the curvature effects do not play essential role in the propagation. Neither narrow isthmuses nor sharp corners of the obstacles, being classical objects for production of extremely curved wave front, affect non-inhibited wave propagation. The curvature-related phenomena of the propagation block and wave detachment from the obstacle boundary were observed only after partial suppression of the sodium channels with Lidocaine. Computer simulations confirmed the experimental observations. The explanation of the observed phenomena refers to the fact that the heart tissue is made of finite size cells so that curvature radii smaller than the cardiomyocyte size loses sense, and in non-inhibited tissue the single cell is capable to transmit excitation to its neighbors.

Photo-Control of Excitation Waves in Cardiomyocyte Tissue Culture

Azobenzene photoswitches were recently reported to control the activity of neural cells and heart beat in leeches. Here, we report photocontrol of excitation of cultured cardiomyocytes that have been made light sensitive by using the addition of azobenzene trimethylammonium bromide (AzoTAB). The trans-isomer of AzoTAB reversibly suppresses spontaneous activity and propagation of excitation waves, whereas the cis-isomer has no detectable effect on the electrical properties of cardiomyocytes. Photoisomerization of AzoTAB was achieved by switching the illumination wavelength, λ, from ~440 nm (trans-isomer) to ~350 nm (cis-isomer). Simultaneous irradiation at two wavelengths with properly chosen intensities allowed for dynamic control of the cis-isomer/trans-isomer ratio and the level of excitability from normal to fully unexcitable. Experiments were conducted by using AzoTAB-treated confluent monolayers of neonatal rat cardiomyocytes. Excitation waves were monitored by using the Ca2+-sensitive fluorescent dye Fluo-4. By projecting two-wavelength illumination patterns onto otherwise uniform cell layers, we were able to create excitable networks with the desired topology, dimensions, and functional properties. The present article discusses potential applications of this technique for the analysis of complex patterns of electrical excitation and cardiac arrhythmias.

Digital photocontrol of the network of live excitable cells

Recent development of tissue engineering techniques allows creating and maintaining almost indefinitely networks of excitable cells with desired architecture. We coupled the network of live excitable cardiac cells with a common computer by sensitizing them to light, projecting a light pattern on the layer of cells, and monitoring excitation with the aid of fluorescent probes (optical mapping). As a sensitizing substance we used azobenzene trimethylammonium bromide (AzoTAB). This substance undergoes cis-trans-photoisomerization and trans-isomer of AzoTAB inhibits excitation in the cardiac cells, while cis-isomer does not. AzoTAB-mediated sensitization allows, thus, reversible and dynamic control of the excitation waves through the entire cardiomyocyte network either uniformly, or in a preferred spatial pattern. Technically, it was achieved by coupling a common digital projector with a macroview microscope and using computer graphic software for creating the projected pattern of conducting pathways. This approach allows real time interactive photocontrol of the heart tissue.

Influence of patterned topographic features on the formation of cardiac cell clusters and their rhythmic activities

In conventional primary cultures, cardiac cells prepared from a newborn rat undergo spontaneous formation of cell clusters after several days. These cell clusters may be non-homogeneously distributed on a flat surface and show irregular beating which can be recorded by calcium ion imaging. In order to improve the cell cluster homogeneity and the beating regularity, patterned topographic features were used to guide the cellular growth and the cell layer formation. On the substrate with an array of broadly spaced cross features made of photoresist, cells grew on the places that were not occupied by the crosses and thus formed a cell layer with interconnected cell clusters. Accordingly, spatially coordinated regular beating could be recorded over the whole patterned area. In contrast, when cultured on the substrate with broadly spaced but inter-connected cross features, the cardiac cell layer showed beatings which were neither coordinated in space nor regular in time. Finally, when cultured on the substrate with narrowly spaced features, the cell beating became spatially coordinated but still remained irregular. Our results suggest a way to improve the rhythmic property of cultured cardiac cell layers which might be useful for further investigations.