Chronic Prestress Regulation of Micro-Heart Muscle Physiology

Abstract

Tissue engineered in-vitro models are an essential tool in biomedical research. Tissue geometry is a key determinant of function, but controlling geometry of micro-scale tissues remains a challenge. We developed a new double molding approach that allows precise replication of high-resolution stereolithographic prints into poly(dimethylsiloxane), facilitating rapid design iterations and highly parallelized sample production. Hydrogels are used as an intermediary mold, and gel mechanical properties including crosslink density predict replication fidelity. We leveraged this approach to study the effects of geometry on the electrophysiology of miniaturized heart muscles engineered from human induced pluripotent stem cells (iPSC). Geometries predicted to increase tissue prestress globally affected cardiomyocyte structure and tissue electrophysiology. Strikingly, pharmacologic studies revealed a prestress threshold is required for sodium channel function. Analysis of RNA and protein levels suggest electrophysiology changes were related to post-transcriptional and potentially post-translational changes of the gap-junction protein Connexin 43 and the sodium channel Nav1.5.

Mechanical Resistance to Micro-Heart Tissue Contractility unveils early Structural and Functional Pathology in iPSC Models of Hypertrophic Cardiomyopathy

“…Sylgard 184 was then molded off the agar gel to get a positive replica of the original 3D print. Finally, the PDMS replicas of the 3D prints were treated with fluorosilane before Sylgard 184 stencils were cast off these replica PDMS molds(32, 80). PDMS was cured against the replica mold under tight compression so that the “dog bone” shapes formed through-holes through the PDMS stencils.…”

“…3) . We used tissue gross shape as our first quality control metric because of our recent findings that overall μHM shape impacts physiology(80). Tissues that deviated significantly from the expected “dog bone” shape or were delaminated from the substrate were excluded.…”

“…Tissue quality was determined by tissue shape, visible cellular death, and maintenance of adhesion to the substrates (SI Figure . 3). We used tissue gross shape as our first quality control metric because of our recent findings that overall µHM shape impacts physiology (80). Tissues that deviated significantly from the expected "dog bone" shape or were delaminated from the substrate were excluded.…”

Dynamic control of contractile resistance to iPSC‐derived micro‐heart muscle arrays

“…To create μHM, "dog bone" shaped stencils were fabricated using the previously described Hydrogel Assisted STereolithographic Elastomer (HASTE) prototyping method. 47 This method allows replication of high resolution, stereolithographic 3D prints into PDMS elastomer. Briefly, initial prints were designed using Autodesk Inventor Professional to have multiple dog bone shaped extrusions with knobs on each end having 1 mm  1 mm square dimensions connected by a 1 mm  200 μm rectangular "shaft" on a 3 mm thick base plate.…”