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Introduction: Cell structure and migration is impacted by the mechanical properties and geometry of the cell adhesive environment. Most studies to date investigating the effects of 3D environments on cells have not controlled geometry at the single-cell level, making it difficult to understand the influence of 3D environmental cues on single cells. Here, we developed microwell platforms to investigate the effects of 2D vs. 3D geometries on single-cell F-actin and nuclear organization. Methods: We used microfabrication techniques to fabricate three polyacrylamide platforms: 3D microwells with a 3D adhesive environment (3D/3D), 3D microwells with 2D adhesive areas at the bottom only (3D/2D), and flat 2D gels with 2D patterned adhesive areas (2D/2D). We measured geometric swelling and Young's modulus of the platforms. We then cultured C2C12 myoblasts on each platform and evaluated the effects of the engineered microenvironments on F-actin structure and nuclear shape. Results: We tuned the mechanical characteristics of the microfabricated platforms by manipulating the gel formulation. Crosslinker ratio strongly influenced geometric swelling whereas total polymer content primarily affected Young's modulus. When comparing cells in these platforms, we found significant effects on F-actin and nuclear structures. Our analysis showed that a 3D/3D environment was necessary to increase actin and nuclear height. A 3D/2D environment was sufficient to increase actin alignment and nuclear aspect ratio compared to a 2D/2D environment. Conclusions: Using our novel polyacrylamide platforms, we were able to decouple the effects of 3D confinement and adhesive environment, finding that both influenced actin and nuclear structure. [ABSTRACT FROM AUTHOR]

Polyacrylamide gels functionalized with extracellular matrix proteins are commonly used as cell culture platforms to evaluate the combined effects of extracellular matrix composition, cell geometry and substrate rigidity on cell physiology. For this purpose, protein transfer onto the surface of polyacrylamide hydrogels must result in geometrically well-resolved micropatterns with homogeneous protein distribution. Yet the outcomes of micropatterning methods have not been pairwise evaluated against these criteria. We report a high-fidelity photoresist lift-off patterning method to pattern ECM proteins on polyacrylamide hydrogels with elastic moduli ranging from 5 to 25 kPa. We directly compare the protein transfer efficiency and pattern geometrical accuracy of this protocol to the widely used microcontact printing method. Lift-off patterning achieves higher protein transfer efficiency, increases pattern accuracy, increases pattern yield, and reduces variability of these factors within arrays of patterns as it bypasses the drying and transfer steps of microcontact printing. We demonstrate that lift-off patterned hydrogels successfully control cell size and shape and enable long-term imaging of actin intracellular structure and lamellipodia dynamics when we culture epithelial cells on these substrates. [ABSTRACT FROM AUTHOR]

A number of technologies are available to increase the abundance of DNA markers and contribute to developing high resolution genetic maps suitable for genetic analysis. The aim of this study was to expand the number of Diversity Array Technology (DArT) markers on the wheat array that can be mapped in the wheat genome, and to determine their chromosomal location with respect to simple sequence repeat (SSR) markers and their position on the cytogenetic map. A total of 749 and 512 individual DArT and SSR markers, respectively, were identified on at least one of four genetic maps derived from recombinant inbred line (RIL) or doubled haploid (DH) populations. A number of clustered DArT markers were observed in each genetic map, in which 20–34% of markers were redundant. Segregation distortion of DArT and SSR markers was also observed in each mapping population. Only 14% of markers on the Version 2.0 wheat array were assigned to chromosomal bins by deletion mapping using aneuploid lines. In this regard, methylation effects need to be considered when applying DArT marker in genetic mapping. However, deletion mapping of DArT markers provides a reference to align genetic and cytogenetic maps and estimate the coverage of DNA markers across the wheat genome. [ABSTRACT FROM AUTHOR]

Human-induced pluripotent stem cell-derived cardiomyocytes are a potentially unlimited cell source and promising patient-specific in vitro model of cardiac diseases. Yet, these cells are limited by immaturity and population heterogeneity. Current in vitro studies aiming at better understanding of the mechanical and chemical cues in the microenvironment that drive cellular maturation involve deformable materials and precise manipulation of the microenvironment with, for example, micropatterns. Such microenvironment manipulation most often involves microfabrication protocols which are time-consuming, require cleanroom facilities and photolithography expertise. Here, we present a method to increase the scale of the fabrication pipeline, thereby enabling large-batch generation of shelf-stable microenvironment protein templates on glass chips. This decreases fabrication time and allows for more flexibility in the subsequent steps, for example, in tuning the material properties and the selection of extracellular matrix or cell proteins. Further, the fabrication of deformable hydrogels has been optimized for compatibility with these templates, in addition to the templates being able to be used to acquire protein patterns directly on the glass chips. With our approach, we have successfully controlled the shapes of cardiomyocytes seeded on Matrigel-patterned hydrogels. [ABSTRACT FROM AUTHOR]