In vivo, cardiomyocytes are exposed to multiple biochemical and physical cues including topographical and electrical cues. During prolonged in vitro cultivation in standard tissue culture set-ups, cardiomyocytes are known to de-differentiate due to the lack of appropriate micro-environmental cues. Most currently available cell culture systems provide only a single biophysical cue, thus development of advanced cell cultivation systems incorporating multiple cues is urgently needed. We report here the development of a microfabricated system, incorporating topographical and electrical cues on a single chip, which enables cultivation of differentiated cardiomyocytes. The cell culture chips were created by hot embossing of polystyrene, to create microgrooves and microridges of precisely defined depth, width and periodicity. Substrates consisting of 0.5 microm-wide grooves and 0.5 microm-wide ridges (1 microm period) and those consisting of 3 microm-wide grooves and 1 microm-wide ridges (4 microm period) were investigated, with smooth surfaces used as controls. The depth of the microgrooves was 400 nm. The two gold electrodes were electrodeposited 1 cm apart such that the microgrooves in-between were oriented either parallel or perpendicular to the electrodes, enabling studies of interaction between topographical and electrical cues. Neonatal rat cardiomyocytes cultivated on microgrooved substrates for 7 days were elongated and aligned along the microgrooves forming a well developed contractile apparatus, as evidenced by sarcomeric alpha-actinin staining, with a more pronounced effect on substrates with 1 microm compared to 4 microm periodicity. Importantly, simultaneous application of biphasic electrical pulses and topographical cues resulted in gap junctions confined to the cell-cell end junctions rather than the punctate distribution found in neonatal cells. Electrical field stimulation further enhanced cardiomyocyte elongation when microgrooves were oriented parallel to the electric field. Due to the compatibility of the described cell culture chips with fluorescence and optical microscopy as well as the ability to independently control field stimulation parameters, biochemical and topographical cues on each chip, this system may in the future become a useful tool in drug development and maturation of cardiomyocytes derived from stem cells.
Background: Arrayed primer extension (APEX) is a microarray-based rapid minisequencing methodology that may have utility in 'personalized medicine' applications that involve genetic diagnostics of single nucleotide polymorphisms (SNPs). However, to date there have been few reports that objectively evaluate the assay completion rate, call rate and accuracy of APEX. We have further developed robust assay design, chemistry and analysis methodologies, and have sought to determine how effective APEX is in comparison to leading 'gold-standard' genotyping platforms. Our methods have been tested against industry-leading technologies in two blinded experiments based on Coriell DNA samples and SNP genotype data from the International HapMap Project.
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