Novel Micropatterned Cardiac Cell Cultures with Realistic Ventricular Microstructure

Abstract

Systematic studies of cardiac structure-function relationships to date have been hindered by the intrinsic complexity and variability of in vivo and ex vivo model systems. Thus, we set out to develop a reproducible cell culture system that can accurately replicate the realistic microstructure of native cardiac tissues. Using cell micropatterning techniques, we aligned cultured cardiomyocytes at micro- and macroscopic spatial scales to follow local directions of cardiac fibers in murine ventricular cross sections, as measured by high-resolution diffusion tensor magnetic resonance imaging. To elucidate the roles of ventricular tissue microstructure in macroscopic impulse conduction, we optically mapped membrane potentials in micropatterned cardiac cultures with realistic tissue boundaries and natural cell orientation, cardiac cultures with realistic tissue boundaries but random cell orientation, and standard isotropic monolayers. At 2 Hz pacing, both microscopic changes in cell orientation and ventricular tissue boundaries independently and synergistically increased the spatial dispersion of conduction velocity, but not the action potential duration. The realistic variations in intramural microstructure created unique spatial signatures in micro- and macroscopic impulse propagation within ventricular cross-section cultures. This novel in vitro model system is expected to help bridge the existing gap between experimental structure-function studies in standard cardiac monolayers and intact heart tissues.

Capillary Force Lithography for Cardiac Tissue Engineering

“…Various other methods, such as microcontact printing, electrospinning, and microtopography, have previously been employed to control the structure of engineered cardiac tissue on the microscale [39][40][41] . While these techniques have proven successful in gaining cellular alignment, it is likely that the structure and function of in vivo cardiac tissue is governed by the much smaller, nanotopographical cues of the ECM and thus our NP substrate should prove advantageous.…”

Multiscale Biomimetic Topography for the Alignment of Neonatal and Embryonic Stem Cell-Derived Heart Cells

“…Cell extraction was performed as previously described with some modifications. 21,25,27 Briefly, hearts were extracted from neonatal mice and placed in chilled 1· PBS (calcium magnesium free). Blood was removed by squeezing the ventricles, rinsed with PBS, and placed in HBSS for 10 min.…”

“…15,20 Fabrication of cell culture substrates that mimic the native environment found in the heart may improve the culture conditions of cells for the development of functional cardiac tissue. [21][22][23][24] The alignment of CM has been studied for the last decade using different microfabrication (e.g., microcontact printing, abrasion, photolithography, hot embossing, electrospinning, and laser ablation) approaches. [25][26][27][28][29][30] Studies have also shown that these microtopographic cues have a greater effect on cell alignment than electrical cues.…”

A Method to Replicate the Microstructure of Heart Tissue In Vitro Using DTMRI-Based Cell Micropatterning

“…3 Briefly, coverslips were stained with a voltage sensitive dye (Di-4 ANEPPS, 16 lmol/L) for 5 min, transferred to a custom, thermo-regulated chamber perfused with Tyrodes solution, and stimulated with a platinum bipolar point electrode. Optical signals were recorded by a 504 optical-fiber photodiode array (RedShirt Imaging).…”

“…Action potential activation times, defined as the times of maximum upstroke, were registered onto the culture's corresponding DTMRI angle map to produce isochrone maps of activation. 3 Local conduction velocities were calculated from activation times and spatially averaged across the entire mapping area as previously described. 3 …”