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Versatile Fabrication of Size- and Shape-Controllable Nanofibrous Concave Microwells for Cell Spheroid Formation.

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.