Although the microfabrication techniques for microwells enabled to guide physiologically relevant three-dimensional cell spheroid formation, there have been substantial interests to more closely mimic nano/microtopographies of in vivo cellular microenvironment. Here, we developed a versatile fabrication process for nanofibrous concave microwells (NCMs) with a controllable size and shape. The key to the fabrication process was the use of an array of hemispherical convex electrolyte solution drops as the grounded collector for electrospinning, which greatly improved the degree of freedom of the size, shape, and curvature of an NCM. A polymer substrate with through-holes was prepared for the electrolyte solution to come out through the hole and to naturally form a convex shape because of surface tension. Subsequent electrolyte-assisted electrospinning process enabled to achieve various arrays of NCMs of triangular, rectangular, and circular shapes with sizes ranging from 1000 μm down to 250 μm. As one example of biomedical applications, the formation of human hepatoma cell line (HepG2) spheroids was demonstrated on the NCMs. The results indicated that the NCM enabled uniform, size-controllable spheroid formation of HepG2 cells, resulting in 1.5 times higher secretion of albumin from HepG2 cells on the NCM on day 14 compared with those on a nanofibrous flat microwell as a control.

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The endothelialization on the poly (ε-caprolactone) nanofiber has been limited due to its low hydrophilicity. The aim of this study was to immobilize collagen on an ultra-thin poly (ε-caprolactone) nanofiber membrane without altering the nanofiber structure and maintaining the endothelial cell homeostasis on it. We immobilized collagen on the poly (ε-caprolactone) nanofiber using hydrolysis by NaOH treatment and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/sulfo- N-hydroxysulfosuccinimide reaction as a cost-effective and stable approach. NaOH was first applied to render the poly (ε-caprolactone) nanofiber hydrophilic. Subsequently, collagen was immobilized on the surface of the poly (ε-caprolactone) nanofibers using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/sulfo- N-hydroxysulfosuccinimide. Scanning electron microscopy, Fourier transform infrared spectroscopy, transmission electron microscopy, and fluorescence microscopy were used to verify stable collagen immobilization on the surface of the poly (ε-caprolactone) nanofibers and the maintenance of the original structure of poly (ε-caprolactone) nanofibers. Furthermore, human endothelial cells were cultured on the collagen-immobilized poly (ε-caprolactone) nanofiber membrane and expressed tight junction proteins with the increase in transendothelial electrical resistance, which demonstrated the maintenance of the endothelial cell homeostasis on the collagen-immobilized-poly (ε-caprolactone) nanofiber membrane. Thus, we expected that this process would be promising for maintaining cell homeostasis on the ultra-thin poly (ε-caprolactone) nanofiber scaffolds.

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