Cell culture chips for simultaneous application of topographical and electrical cues enhance phenotype of cardiomyocytes

Abstract

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.

Mesenchymal stem cell mechanobiology and emerging experimental platforms

“…For example, neonatal rat cardiomyocytes that were cultivated on micro-grooved substrates for 7 days were elongated and aligned along the microgrooves forming a well-developed contractile apparatus, as evidenced by sarcomeric a-actinin staining (figure 6c) [140]. Simultaneous application of biphasic electrical pulses and topographical cues resulted in gap junctions that were confined to the cell-cell end junctions rather than to the punctate distribution found in neonatal cells, and electrical field stimulation further enhanced cardiomyocyte elongation when microgrooves were oriented parallel to the electric field.…”

“…Mechanical cues are used in combination with electrical and biochemical stimulation to study cardiovascular constructs in organ-on-a-chip microdevices [76,140]. For example, neonatal rat cardiomyocytes that were cultivated on micro-grooved substrates for 7 days were elongated and aligned along the microgrooves forming a well-developed contractile apparatus, as evidenced by sarcomeric a-actinin staining (figure 6c) [140].…”

“…'Organ-on-a-chip' platforms that recapitulate key aspects of in vivo niches. (a) A 'lung-on-a-chip' device that uses compartmentalized PDMS microchannels to form an alveolar -capillary barrier on a thin, porous, flexible PDMS membrane coated with ECM and recreates physiological breathing movements by applying vacuum to the side chambers that cause mechanical stretching of the PDMS membrane, reproduced from [75] with permission from AAAS; (b) a multi-layer microfluidic device for efficient culture and analysis of renal tubular cells, reproduced with from [139] with permission from the Royal Society of Chemistry; (c) schematic and photograph of a cardiomyocyte culture chip with microgrooves oriented either parallel or perpendicular to electrodes, reproduced from [140] with permission from the Royal Society of Chemistry; (d ) 'heart-on-a-chip' for contractility assays with anisotropic layers of myocytes, scale bar, 20 mm, reproduced from [76] with permission from the Royal Society of Chemistry.…”

Structural and functional plasticity in long-term cultures of adult ventricular myocytes

“…One of simplest approaches in this regard has been the application of electrical stimulation to cardiomyocytes in culture, which has been reported to engender some enhancement of conduction and myofibrillar organization in other systems [34, 43, 48, 62, 63]. Here, we applied such stimulation to aGPVM cultures, commencing just after monolayer confluency was achieved (10–14 days), and continuing through 21 days in culture.…”

Spatially and temporally controlled hydrogels for tissue engineering

“…In addition to the dynamic mechanical stimuli, electrical stimulation can also influence cell behaviors and functions, such as proliferation, differentiation, and maturation [366, 367]. Particularly, the electrical signaling is regarded as one of the most crucial microenvironmental cues to direct neuronal cell functions.…”