Small-artery-mimicking multi-layered 3D co-culture in a self-folding porous graphene-based film.

In vitro vessel-mimicking models have been spotlighted as a powerful tool for investigating cellular behaviours in vascular development and diseases. However, it is still challenging to create micro-scale tubular tissues while mimicking the structural features of small arteries. Here, we propose a 3D culture model of small vascular tissue using a self-folding graphene-based porous film. Vascular endothelial cells were encapsulated within the self-folding film to create a cellular construct with a controlled curvature radius ranging from 10 to 100 µm, which is comparable to the size of a human arteriole. Additionally, vascular endothelial cells and smooth muscle cells were separately co-cultured on the inner and outer surfaces of the folded film, respectively. The porous wall worked as a permeable barrier between them, affecting the cell-cell communications like the extracellular layer in the artery wall. Thus, the culture model recapitulates the structural features of a small artery and will help us better understand intercellular communications at the artery wall in physiological and pathological conditions.

Observation of intracellular protein localization area in a single neuron using gold nanoparticles with a scanning electron microscope.

The localization areas of intracellular proteins in rat cortical neurons were visualized using a scanning electron microscope (SEM) coupled with a focused ion beam (FIB) system. To obtain a clear contrast in the SEM images, gold nanoparticles (GNPs) were bound to specific intracellular proteins by antigen-antibody reactions. By obtaining a cross section of the desired location of the neurons by FIB milling under the SEM imaging condition, it was possible to observe the proteins inside the cells as clear bright spots. When a neuron was stained with anti-tau and anti-histone H1 antibodies, the bright spots were localized in the cross section of the axon and the nucleus, respectively. It was confirmed that targeted proteins in a single neuron on a substrate could be successfully identified. The development of FIB/SEM observation with immunological GNP staining will offer important information for the stable growth of neurons on various substrate structures, since the elongation and turning of axons on the substrates are activated by the redistribution of intracellular proteins.

Self-Folded Three-Dimensional Graphene with a Tunable Shape and Conductivity.

Three-dimensional (3D) graphene architectures are of great interest as applications in flexible electronics and biointerfaces. In this study, we demonstrate the facile formation of predetermined 3D polymeric microstructures simply by transferring monolayer graphene. The graphene adheres to the surface of polymeric films via noncovalent π-π stacking bonding and induces a sloped internal strain, leading to the self-rolling of 3D microscale architectures. Micropatterns and varied thicknesses of the 2D films prior to the self-rolling allows for control over the resulting 3D geometries. The strain then present on the hexagonal unit cell of the graphene produces a nonlinear electrical conductivity across the device. The driving force behind the self-folding process arises from the reconfiguration of the molecules within the crystalline materials. We believe that this effective and versatile way of realizing a 3D graphene structure is potentially applicable to alternative 2D layered materials as well as other flexible polymeric templates.

Scanning Electron Microscopy Observation of Interface Between Single Neurons and Conductive Surfaces.

Interfaces between single neurons and conductive substrates were investigated using focused ion beam (FIB) milling and subsequent scanning electron microscopy (SEM) observation. The interfaces play an important role in controlling neuronal growth when we fabricate neuron-nanostructure integrated devices. Cross sectional images of cultivated neurons obtained with an FIB/SEM dual system show the clear affinity of the neurons for the substrates. Very few neurons attached themselves to indium tin oxide (ITO) and this repulsion yielded a wide interspace at the neuron-ITO interface. A neuron-gold interface exhibited partial adhesion. On the other hand, a neuron-titanium interface showed good adhesion and small interspaces were observed. These results are consistent with an assessment made using fluorescence microscopy. We expect the much higher spatial resolution of SEM images to provide us with more detailed information. Our study shows that the interface between a single neuron and a substrate offers useful information as regards improving surface properties and establishing neuron-nanostructure integrated devices.