A Method to Replicate the Microstructure of Heart Tissue <Emphasis Type="Italic">In Vitro</Emphasis> Using DTMRI-Based Cell Micropatterning

A novel cell culture methodology is described in which diffusion tensor magnetic resonance imaging (DTMRI) and cell micropatterning are combined to fabricate cell monolayers that replicate realistic cross-sectional tissue structure. As a proof-of-principle, neonatal rat ventricular myocyte (NRVM) monolayers were cultured to replicate the tissue microstructure of murine ventricular cross-sections. Specifically, DTMRI-measured in-plane cardiac fiber directions were converted into soft-lithography photomasks. Silicone stamps fabricated from the photomasks deposit fibronectin patterns to guide local cellular alignment. Fibronectin patterns consisted of a matrix of 190 μm² subregions, each comprised of parallel lines 11–20 μm-wide, spaced 2–8.5 μm apart, and angled to match local DTMRI-measured fiber directions. Within 6 days of culture, NRVMs established confluent, electrically coupled monolayers, and for 18 μm-wide, 5 μm-spaced lines, directions of cell alignment in subregions microscopically replicated DTMRI-measurements with a local error of 7.2 ± 4.1°. By adjusting fibronectin line widths and spacings, cell elongation, gap junctional membrane distribution, and local cellular disarray were altered without changing the dominant directions of cell alignment in individual subregions. Changes in the anisotropy of electrical propagation were assessed by optically mapping membrane potentials. This novel methodology is expected to enable systematic studies of intramural structure–function relationships in both healthy and structurally remodeled hearts.