In Vivo-Like Morphology of Intercalated Discs Achieved in a Neonatal Cardiomyocyte Culture Model.

In vitro cultures to be used in various analytical investigations of cardiomyocyte (CM) growth and function for enhancing insight into physiological and pathological mechanisms should closely express in vivo morphology. The aim of the studies is to explore how to use microfabrication and physical-cue-addition techniques to establish a neonatal rat CM culture model that expresses an end-to-end connected rod shape with in vivo-like intercalated discs (ICDs). Freshly isolated neonatal rat CMs were cultured on microgrooved polydimethylsiloxane substrate. Cell alignment and ICD orientation were evaluated using confocal fluorescence and transmission electron microscopy under various combinations of different culture conditions. Cyclic stretch and blebbistatin tests were conducted to explore mechanical and electrical effects. Laboratory-made MATLAB software was developed to quantify cell alignment and ICD orientation. Our results demonstrate that the mechanical effect associated with the electrical stimulation may contribute to step-like ICD formation viewed from the top. In addition, our study reveals that a suspended elastic substrate that was slack with scattered folds, not taut, enabled CM contraction of equal strength on both apical and basal cell surfaces, allowing the cultured CMs to express a three-dimensional rod shape with disc-like ICDs viewed cross-sectionally. Impact statement In this article, we describe how the tugging forces generated by cardiomyocytes (CMs) facilitate the formation of the morphology of the intercalated discs (ICDs) to achieve mechanoelectrical coupling between CMs. Correspondingly, we report experimental techniques we developed to enable the in vivo-like behavior of the tugging forces to support the development of in vivo-like morphology in ICDs. These techniques will enhance insight into physiological and pathological mechanisms related to the development of tissue-engineered cardiac constructs in various analytical investigations of CM growth and function.

Silicon nanowire-induced maturation of cardiomyocytes derived from human induced pluripotent stem cells.

The current inability to derive mature cardiomyocytes from human pluripotent stem cells has been the limiting step for transitioning this powerful technology into clinical therapies. To address this, scaffold-based tissue engineering approaches have been utilized to mimic heart development in vitro and promote maturation of cardiomyocytes derived from human pluripotent stem cells. While scaffolds can provide 3D microenvironments, current scaffolds lack the matched physical/chemical/biological properties of native extracellular environments. On the other hand, scaffold-free, 3D cardiac spheroids (i.e., spherical-shaped microtissues) prepared by seeding cardiomyocytes into agarose microwells were shown to improve cardiac functions. However, cardiomyocytes within the spheroids could not assemble in a controlled manner and led to compromised, unsynchronized contractions. Here, we show, for the first time, that incorporation of a trace amount (i.e., ∼0.004% w/v) of electrically conductive silicon nanowires (e-SiNWs) in otherwise scaffold-free cardiac spheroids can form an electrically conductive network, leading to synchronized and significantly enhanced contraction (i.e., >55% increase in average contraction amplitude), resulting in significantly more advanced cellular structural and contractile maturation.