ECS-0335765. == Contributor Information == Jingwei Xie, Department of Biomedical Engineering, Washington University, St. nanoscale structural cues.[710] In contrast to the aforementioned techniques, electrospinning is a simpler and more versatile nanofabrication technique that can be used to process many types of materials into long fibers with diameters ranging from tens of nanometers to several micrometers.[11] Owing to the high ratio of surface area to volume for a nanofiber and the one-dimensional morphology, electrospun nanofibers can mimic the architecture of the extracellular matrix CB-6644 (ECM), making them attractive for a broad range of biomedical applications.[10] Electrospun fibers are typically deposited on a conductive collector as a random, nonwoven mat. Several techniques have been developed to control the alignment and organization of the nanofibers. For example, electrospun fibers can be aligned into uniaxial arrays by manipulating the electric field or by applying a mechanical force.[13,14] However, little effort has been devoted to the fabrication of membranes with complex surface structures via electrospinning. One recent report described the use of a wire mesh and an array of square-shaped protrusions made of iron as collectors for the fabrication of nanofiber membranes with patterned and woven structures.[15] Another study demonstrated the use of two-dimensional arrays of pins as collectors to generate mesh-like nanofiber CB-6644 membranes over large areas.[16] The nanofiber membranes described in these reports, however, were essentially flat films with no microwells on the surface (limiting their potential as cell microarray substrates) and no biological applications was demonstrated. Here we describe a new approach based on electrospinning for generating arrayed microwells on a nanofiber membrane whose surface also contains structural cues in the form of uniaxially aligned nanoscale features. Compared to the conventional microwells for cell culture, which are commonly used as individual, isolated containers, the arrayed microwells presented here could be used both individually and collectively. Specifically, with an array of stainless steel beads as the collectors for electrospinning, we demonstrated the fabrication of poly(-caprolactone) (PCL) nanofiber membranes with arrayed microwells and controllable structural cues on the surface. We further demonstrated two applications for this novel class of nanofiber scaffolds:i) formation of cell microarrays; andii) formation of neuronal networks. The experimental setup is similar to what we used in previous studies with the exception of a new design for CB-6644 the collector (Figure 1a),[1719] which can be easily assembled from stainless steel beads and reconfigured into different patterns.Figure 1bshows the distribution of electric field between the needle tip and the arrayed metal beads, obtained using the software COMSOL 3.3 (COMSOL Inc, Burlington, MA). Note that the electric field vectors above each bead point directly towards the surface of the bead, similar to a conventional collector. However, the electric field vectors above the gap region between two adjacent beads are split into two main streams, pointing towards each bead. This pattern suggests that the nanofibers deposited directly onto the beads will be randomly oriented while those deposited across the gap between adjacent beads will be uniaxially aligned.Figure 2ashows a photograph of a nanofiber membrane collected with a close packed array of stainless steel beads 2 mm in diameter.Figure 2bshows a typical scanning electron microscopy (SEM) image of the same membrane, illustrating a complex architecture composed of a hexagonal array of microwells interconnected through a network of uniaxially aligned nanofibers. The depth of the wells was 430 8 m, as measured by analyzing cross-sectional images of the membrane. When the collectors were constructed from stainless steel beads of 1 1 mm in diameter, the depth was reduced to 219 6 m.Figure 2, cf, shows SEM images of the regions indicated inFigure 2bat a higher magnification. It is clear that the nanofibers deposited on the surface of stainless steel beads were randomly oriented whereas those deposited across the gap between two adjacent beads were uniaxially aligned. There was also a short transition zone from random to uniaxial alignment.Figure 2fshows that the density of fibers was much lower across the void among three neighboring beads than other regions of the membrane and the fibers deposited in the void region were randomly distributed. == Figure 1. == (a) A schematic illustrating the ATN1 electrospinning setup used for fabricating a nanofiber membrane with arrayed microwells and aligned structural cues on the surface, and (b) a simulated distribution of electric field vectors in the region between the needle and the collector. == Figure 2. == (a) Optical micrographs and (bf) SEM images of a membrane composed of PCL electrospun nanofibers. The inset in (a) shows an evidence for the formation of microwells on the membrane, as indicated by the shadows. This.