RESUMEN
In this study, we report nanopatterned Nafion microelectrode arrays for in vitro cardiac electrophysiology. With the aim of defining sophisticated Nafion nanostructures with highly ionic conductivity, fabrication parameters such as Nafion concentration and curing temperature were optimized. By increasing curing temperature and Nafion concentration, we were able to control the replication fidelity of Nafion nanopatterns when copied from a PDMS master mold. We also found that cross-sectional morphology and ion current density of nanopatterned Nafion strongly depends on the fabrication parameters. To investigate this dependency, current-voltage analysis was conducted using organic electrochemical transistors (OECT) overlaid with patterned Nafion substrates. Nanopatterned Nafion was found to allow higher ion current densities than unpatterned surfaces. Furthermore, higher curing temperatures were found to render Nafion layers with higher ion/electrical transfer properties. To optimize nanopattern dimensions, electrical current flows, and film uniformity, a final configuration consisting of 5% nanopatterned Nafion cured at 65°C was chosen. Multielectrode arrays (MEAs) were then covered with optimized Nafion nanopatterns and used for electrophysiological analysis of two types of induced pluripotent stem cell-derived cardiomyocytes (iPSCs-CMs). These data highlight the suitability of nanopatterned Nafion, combined with MEAs, for enhancing the cellular environment of iPSC-CMs for use in electrophysiological analysis in vitro.
RESUMEN
Adrenergic receptors are mediators of adrenergic and noradrenergic modulation throughout the brain. Previous studies have provided evidence for the expression of adrenergic receptors in the midbrain auditory nucleus, the inferior colliculus (IC), but have not examined the cellular patterns of expression in detail. Here, we utilize multichannel fluorescent in situ hybridization to detect the expression of adrenergic receptor-encoding mRNA in the inferior colliculus of male and female mice. We found expression of α1 , α2A , and ß2 receptor-encoding mRNA throughout all areas of the IC. While we observed similar levels of expression of α1 receptor-encoding mRNA across the subregions of the IC, α2A and ß2 receptor-encoding mRNA was expressed differentially. To account for developmental changes in noradrenergic receptor expression, we measured expression levels in mice aged P15, P20, and P60. We observed little change in levels of expression across these ages. To ascertain the modulatory potential of multiple adrenergic receptor subtypes in a single IC cell, we measured co-expression of α1 , α2A , and ß2 receptor-encoding mRNA. We found greater proportions of cells in the IC that expressed no adrenergic receptor-encoding mRNA, α1 and α2A adrenergic receptor-encoding mRNA, and α1, α2A, and ß2 receptor-encoding mRNA than would be predicted by independent expression of each receptor subtype. These data suggest a coordinated pattern of adrenergic receptor expression in the IC and provide the first evidence for adrenergic receptor expression and co-expression in the subregions of the mouse auditory midbrain.
Asunto(s)
Colículos Inferiores/metabolismo , ARN Mensajero/análisis , Receptores Adrenérgicos/metabolismo , Animales , Femenino , Masculino , Ratones , Receptores Adrenérgicos/análisisRESUMEN
Tissue engineering aims to capture the structural and functional aspects of diverse tissue types in vitro. However, most approaches are limited in their ability to produce complex 3D geometries that are essential for tissue function. Tissues, such as the vasculature or chambers of the heart, often possess curved surfaces and hollow lumens that are difficult to recapitulate given their anisotropic architecture. Cell-sheet engineering techniques using thermoresponsive substrates provide a means to stack individual layers of cells with spatial control to create dense, scaffold-free tissues. In this study, we developed a novel method to fabricate complex 3D structures by layering multiple sheets of aligned cells onto flexible scaffolds and casting them into hollow tubular geometries using custom molds and gelatin hydrogels. To enable the fabrication of 3D tissues, we adapted our previously developed thermoresponsive nanopatterned cell-sheet technology by applying it to flexible substrates that could be folded as a form of tissue origami. We demonstrated the versatile nature of this platform by casting aligned sheets of smooth and cardiac muscle cells circumferentially around the surfaces of gelatin hydrogel tubes with hollow lumens. Additionally, we patterned skeletal muscle in the same fashion to recapitulate the 3D curvature that is observed in the muscles of the trunk. The circumferential cell patterning in each case was maintained after one week in culture and even encouraged organized skeletal myotube formation. Additionally, with the application of electrical field stimulation, skeletal myotubes began to assemble functional sarcomeres that could contract. Cardiac tubes could spontaneously contract and be paced for up to one month. Our flexible cell-sheet engineering approach provides an adaptable method to recapitulate more complex 3D geometries with tissue specific customization through the addition of different cell types, mold shapes, and hydrogels. By enabling the fabrication of scaled biomimetic models of human tissues, this approach could potentially be used to investigate tissue structure-function relationships, development, and maturation in the dish.
Asunto(s)
Hidrogeles , Ingeniería de Tejidos , Anisotropía , Gelatina , Humanos , Fibras Musculares Esqueléticas , Andamios del TejidoRESUMEN
Recent advances in stem cell biology and tissue engineering have laid the groundwork for building complex tissues in a dish. We propose that these technologies are ready for a new challenge: recapitulating cardiac morphogenesis in vitro. In development, the heart transforms from a simple linear tube to a four-chambered organ through a complex process called looping. Here, we re-examine heart tube looping through the lens of an engineer and argue that the linear heart tube is an advantageous starting point for tissue engineering. We summarize the structures, signaling pathways, and stresses in the looping heart, and evaluate approaches that could be used to build a linear heart tube and guide it through the process of looping.